Systems and Devices for High-Throughput Sequencing with Semiconductor-Based Detection

ABSTRACT

In one embodiment, a sample surface of a biosensor includes pixel areas and holds a plurality of clusters during a sequence of sampling events such that the clusters are distributed unevenly over the pixel areas. In another embodiment, a biosensor has a sample surface that includes pixel areas and an array of wells overlying the pixel areas, the biosensor including two wells and two clusters per pixel area. The two wells per pixel area include a dominant well and a subordinate well. The dominant well has a larger cross section over the pixel area than the subordinate well. In yet another embodiment, an illumination system is coupled to a biosensor that illuminates the pixel areas with different angles of illumination during a sequence of sampling events, including, for a sampling event, illuminating each of the wells with off-axis illumination to produce asymmetrically illuminated well regions in each of the wells.

PRIORITY APPLICATIONS

This application claims priority as a continuation of U.S. applicationSer. No. 16/241,905 filed Jan. 7, 2019, which claims the benefit of U.S.Provisional Patent Application No. 62/614,934, entitled “SYSTEMS ANDDEVICES FOR HIGH-THROUGHPUT SEQUENCING WITH SEMICONDUCTOR-BASEDDETECTION,” filed on Jan. 8, 2018, (Atty. Docket No. IP-1656-PRV), U.S.Provisional Patent Application No. 62/614,930, entitled “HIGH-THROUGHPUTSEQUENCING WITH SEMICONDUCTOR-BASED DETECTION,” filed on Jan. 8, 2018,(Atty. Docket No. ILLM 1003-1/IP-1653-PRV); and Netherlands ApplicationNo. 2020758, entitled “HIGH-THROUGHPUT SEQUENCING WITHSEMICONDUCTOR-BASED DETECTION,” filed on Apr. 12, 2018, (Atty. DocketNo. IP-1653-NL).

The priority applications are hereby incorporated by reference for allpurposes.

CROSS-REFERENCE TO OTHER APPLICATIONS

The following patent applications are incorporated herein in theirentirety for all purposes:

U.S. Nonprovisional patent application Ser. No. 16/241,902, entitled“HIGH-THROUGHPUT SEQUENCING WITH SEMICONDUCTOR-BASED DETECTION,” filedJan. 7, 2019, (Atty. Docket No. IP-1653-US);

U.S. Provisional Patent Application No. 62/614,690, entitled“MULTIPLEXING OF AN ACTIVE SENSOR DETECTOR USING STRUCTUREDILLUMINATION,” filed on Jan. 8, 2018, (Atty. Docket No. IP-1623-PRV);

U.S. Nonprovisional patent application Ser. No. 13/833,619, entitled“BIOSENSORS FOR BIOLOGICAL OR CHEMICAL ANALYSIS AND SYSTEMS AND METHODSFOR SAME,” filed on Mar. 15, 2013, (Atty. Docket No. IP-0626-US);

U.S. Nonprovisional patent application Ser. No. 15/175,489, entitled“BIOSENSORS FOR BIOLOGICAL OR CHEMICAL ANALYSIS AND METHODS OFMANUFACTURING THE SAME,” filed on Jun. 7, 2016, (Atty. Docket No.IP-0689-US);

U.S. Nonprovisional patent application Ser. No. 13/882,088, entitled“MICRODEVICES AND BIOSENSOR CARTRIDGES FOR BIOLOGICAL OR CHEMICALANALYSIS AND SYSTEMS AND METHODS FOR THE SAME,” filed on Apr. 26, 2013,(Atty. Docket No. IP-0462-US); and

U.S. Nonprovisional patent application Ser. No. 13/624,200, entitled“METHODS AND COMPOSITIONS FOR NUCLEIC ACID SEQUENCING,” filed on Sep.21, 2012, (Atty. Docket No. IP-0538-US).

FIELD OF THE TECHNOLOGY DISCLOSED

Embodiments of the technology disclosed relate generally to sequencingwith CMOS-based detection and more particularly to systems and methodsfor increasing throughput of sequencing with CMOS-based detection.

BACKGROUND

Various protocols in biological or chemical research involve performinga large number of controlled reactions on local support surfaces orwithin predefined reaction chambers (or wells). The desired reactionsmay then be observed or detected and subsequent analysis may helpidentify or reveal properties of chemicals involved in the reaction. Forexample, in some multiplex assays, an unknown analyte (e.g., clusters ofclonally amplified nucleic acids) having an identifiable label (e.g.,fluorescent label) may be exposed to thousands of known probes undercontrolled conditions. Each known probe may be deposited into acorresponding well of a microplate or flow cell. Observing any chemicalreactions that occur between the known probes and the unknown analytewithin the wells may help identify or reveal properties of the analyte.Other examples of such protocols include known DNA sequencing processes,such as sequencing-by-synthesis (SBS) or cyclic-array sequencing.

In some conventional fluorescent-detection protocols, an optical systemis used to direct an excitation light onto fluorescently-labeledanalytes and to also detect the fluorescent signals that may emit fromthe analytes. However, such optical systems can be relatively expensiveand require a larger benchtop footprint. For example, the optical systemmay include an arrangement of lenses, filters, and light sources. Inother proposed detection systems, the controlled reactions occurimmediately over a solid-state imager (e.g., charged-coupled device(CCD) or a complementary metal-oxide-semiconductor (CMOS) sensor) thatdoes not require a large optical assembly to detect the fluorescentem1ss10ns.

However, the proposed solid-state imaging systems may have somelimitations. For example, the solid-state imagers are limited to onecluster base call per sensor (or pixel) and their throughput isdependent on the pixel density of the sensors, which is a function ofthe pixel pitch. Since there are limitations on significantly decreasingthe pixel pitch, it becomes desirable to explore other solutions forincreasing the throughput of solid-state imagers.

An opportunity arises to increase the throughput of solid-state imagingsystems by base calling multiple clusters per sensor (or pixel) and toprovide systems and devices that facilitate the multiple cluster basecall per sensor (or pixel).

Embodiments of the present disclosure relate generally to biological orchemical analysis and more particularly to systems and methods usingdetection devices for biological or chemical analysis.

Various protocols in biological or chemical research involve performinga large number of controlled reactions on local support surfaces orwithin predefined reaction chambers. The desired reactions may then beobserved or detected and subsequent analysis may help identify or revealproperties of chemicals involved in the reaction. For example, in somemultiplex assays, unknown analytes having identifiable labels (e.g.,fluorescent labels) may be exposed to thousands of known probes undercontrolled conditions. Each known probe may be deposited into acorresponding location on a surface. Observing any chemical reactionsthat occur between the known probes and the unknown analyte on thesurface may help identify or reveal properties of the analyte. Otherexamples of such protocols include known DNA sequencing processes, suchas sequencing-by-synthesis (SBS) or cyclic-array sequencing.

In some conventional fluorescent-detection protocols, an optical systemis used to direct an excitation light onto fluorescently-labeledanalytes and to also detect the fluorescent signals that may emit fromthe analytes. The throughput of standard imaging techniques isconstrained by the number of pixels available in the detection device,among other things. As such, these optical systems can be relativelyexpensive and require a relatively large bench-top footprint whendetecting surfaces having large collections of analytes. For example,nucleic acid arrays used in genotyping, expression, or sequencinganalyses can require detection of millions of different sites on thearray per square centimeter. Limits in throughput increase cost anddecrease accuracy of these analyses.

Thus, there exists a need for higher throughput apparatus and methods,for example, to detect nucleic acid arrays. The present disclosureaddresses this need and provides other advantages as well.

BRIEF DESCRIPTION OF THE TECHNOLOGY DISCLOSED

In accordance with one embodiment, a device for base calling is providedthat comprises a receptacle configured to hold a biosensor. Thebiosensor has (a) a sample surface that holds a plurality of clustersduring a sequence of sampling events, (b) an array of sensors configuredto generate a plurality of sequences of pixel signals, and (c) acommunication port which outputs the plurality of sequences of pixelsignals. The array has a number N of active sensors and the sensors inthe array are disposed relative to the sample surface to generaterespective pixel signals during the sequence of sampling events from thenumber N of corresponding pixel areas of the sample surface to producethe plurality of sequences of pixel signals. The device furthercomprises a signal processor coupled to the receptacle. The signalprocessor is configured to receive and to process the plurality ofsequences of pixel signals to classify results of the sequence ofsampling events on clusters in the plurality of clusters, includingusing the plurality of sequences of pixel signals to classify results ofthe sequence of sampling events on a number N+M of clusters in theplurality of clusters from the number N of active sensors, where Mis apositive integer.

The results of the sequence of sampling events can correspond tonucleotide bases in the clusters.

The sampling events can comprise two illumination stages in timesequence, and sequences of pixel signals in the plurality of sequencesof pixel signals can include a set of signal samples for each samplingevent, the set including at least one pixel signal from each of the twoillumination stages.

The signal processor can include logic to classify results for twoclusters from the sequences of pixel signals from a single sensor in thearray of sensors. The logic to classify results for two clusters caninclude mapping a first pixel signal of the set of signal samples for asampling event from a particular sensor into at least four bins, andmapping a second pixel signal of the set of signal samples for thesampling event into at least four bins, and logically combining themapping of the first and second pixel signals to classify the resultsfor two clusters.

The sensors in the array of sensors can comprise light detectors.

The sampling events can comprise two illumination stages in timesequence, and sequences of pixel signals in the plurality of sequencesof pixel signals can include a set of signal samples for each samplingevent, the set including at least one pixel signal from each of the twoillumination stages. The first illumination stage can induceillumination from a given cluster indicating nucleotide bases A and Tand the second illumination stage can induce illumination from a givencluster indicating nucleotide bases C and T, and said classifyingresults can comprise calling one of the nucleotide bases A, C, T or G.

Clusters can be distributed unevenly over the pixel areas of the samplesurface, and the signal processor can execute time sequence and spatialanalysis of the plurality of sequences of pixel signals to detectpatterns of illumination corresponding to individual clusters on thesample surface, and to classify the results of the sampling events forthe individual clusters. The plurality of sequences of pixel signalsencodes differential crosstalk between at least two clusters resultingfrom their uneven distribution over the pixel areas.

The sample surface can comprise an array of wells overlying the pixelareas, including two wells per pixel area, the two wells per pixel areacan include a dominant well and a subordinate well, the dominant wellcan have a larger cross section over the pixel area than the subordinatewell.

The sample surface can comprise an array of wells overlying the pixelareas, and the sampling events can include at least one chemical stagewith a number K of illumination stages where K is a positive integer.The illumination stages of the K illumination stages can illuminate thepixel areas with different angles of illumination, and the sequences ofpixel signals can include a set of signal samples for each samplingevent, the set including the number K of pixel signals for the at leastone chemical stage of the sampling events.

The sample surface can comprise an array of wells overlying the pixelareas, and the sampling events can include a first chemical stage with anumber K of illumination stages where K is a positive integer. Theillumination stages of the K illumination stages can illuminate thepixel areas with different angles of illumination, and a second chemicalstage with a number J of illumination stages where J is a positiveinteger. The illumination stages of the K illumination stages in thefirst chemical stage and of the J illumination stages in the secondchemical stage can illuminate the wells in the array of wells withdifferent angles of illumination, and the sequences of pixel signals caninclude a set of signal samples for each sampling event, the setincluding the number K of pixel signals for the first chemical stageplus the number J of pixel signals for the second chemical stage of thesampling events.

In another embodiment, a biosensor for base calling is provided. Thebiosensor comprises a sampling device. The sampling device includes asample surface that has an array of pixel areas and a solid-state imagerthat has an array of sensors. Each sensor generates pixel signals ineach base calling cycle. Each pixel signal represents light gatheredfrom a corresponding pixel area of the sample surface. The biosensorfurther comprises a signal processor configured for connection to thesampling device. The signal processor receives and processes the pixelsignals from the sensors for base calling in a base calling cycle, anduses the pixel signals from fewer sensors than a number of clusters basecalled in the base calling cycle.

A pixel area can receive light from a well on the sample surface and thewell can hold more than one cluster during the base calling cycle.

A cluster can comprise a plurality of single-stranded deoxyribonucleicacid (abbreviated DNA) fragments that have an identical nucleic acidsequence.

In another embodiment, a method of base calling is provided. For a basecalling cycle of a sequencing by synthesis (abbreviated SBS) run, themethod includes detecting: (1) a first pixel signal that representslight gathered from a first pixel area during a first illumination stageof the base calling cycle and (2) a second pixel signal that representslight gathered from the first pixel area during a second illuminationstage of the base calling cycle. The first pixel area underlies aplurality of clusters that shares the first pixel area. The methodincludes using a combination of the first and second pixel signals toidentify nucleotide bases incorporated onto each cluster of theplurality of clusters during the base calling cycle.

The method can also include mapping the first pixel signal into at leastfour bins and mapping the second pixel signal into at least four bins,and combining the mapping of the first and second pixel signals toidentify the incorporated nucleotide bases.

The method can also include applying the method to identify thenucleotide bases incorporated onto the plurality of clusters at aplurality of pixel areas during the base calling cycle.

The method can also include repeating the method over successive basecalling cycles to identify the nucleotide bases incorporated onto theplurality of clusters at the plurality of pixel areas during each of thebase calling cycles.

For each of the base calling cycles, the method can also includedetecting and storing the first and second pixel signals emitted by theplurality of clusters at the plurality of pixel areas, and after thebase calling cycles, using the combination of the first and second pixelsignals to identify the nucleotide bases incorporated onto the pluralityof clusters at the plurality of pixel areas during each of the previousbase calling cycles.

The first pixel area can receive light from an associated well on asample surface. The first pixel area can receive light from more thanone associated well on the sample surface. The first and second pixelsignals can be gathered by a first sensor from the first pixel area. Thefirst and second pixel signals can be detected by a signal processorconfigured for processing pixel signals gathered by the first sensor.The first illumination stage can induce illumination from the first andsecond clusters to produce emissions from labeled nucleotide bases A andT and the second illumination stage can induce illumination from thefirst and second clusters to produce emissions from labeled nucleotidebases C and T.

In another embodiment, a method of identifying pixel areas with morethan one cluster on a sample surface of a biosensor and base callingclusters at the identified pixel areas is provided. The method includesperforming a plurality of base calling cycles, each base calling cyclehaving a first illumination stage and a second illumination stage. Themethod includes capturing at a sensor associated with a pixel area ofthe sample surface, (1) a first set of intensity values generated duringthe first illumination stage of the base calling cycles, and (2) asecond set of intensity values generated during the second illuminationstage of the base calling cycles. The method includes fitting sixteendistributions to the first and second sets of intensity values using asignal processor and, based on the fitting, classifying the pixel areaas having more than one cluster. For a successive base calling cycle,the method includes detecting the first and second sets of intensityvalues for a cluster group at the pixel area using the signal processor,and selecting a distribution for the cluster group. The distributionidentifies a nucleotide base present in each cluster of the clustergroup.

The method can include fitting comprises using one or more algorithms,including a k-means clustering algorithm, a k-means-like clusteringalgorithm, an expectation maximization algorithm, and a histogram basedalgorithm.

The method can include normalizing the intensity values.

The pixel area can receive light from an associated well on the samplesurface.

In another embodiment, a device for base calling is provided. The devicecomprises a receptacle configured to hold a biosensor. The biosensor hasa sample surface. The sample surface includes pixel areas that underlaya plurality of clusters during a sequence of sampling events such thatthe clusters are distributed unevenly over the pixel areas. Thebiosensor also has an array of sensors configured to generate aplurality of sequences of pixel signals. The array has a number N ofactive sensors. The sensors in the array are disposed relative to thesample surface to generate respective pixel signals during the sequenceof sampling events from the number N of corresponding pixel areas of thesample surface to produce the plurality of sequences of pixel signals.The biosensor also has a communication port which outputs the pluralityof sequences of pixel signals. The device further comprises a signalprocessor coupled to the receptacle. The signal processor is configuredto execute time sequence and spatial analysis of the plurality ofsequences of pixel signals to detect patterns of illuminationcorresponding to a number N+M of individual clusters on the samplesurface from the number N of active sensors, where M is a positiveinteger, and to classify the results of the sequence of sampling eventsfor the number N+M of individual clusters. The plurality of sequences ofpixel signals encodes differential crosstalk between at least twoclusters resulting from their uneven distribution over the pixel areas.

The signal processor can use the detected patterns of illumination tolocate the number N+M of individual clusters on the sample surface fromthe number N of active sensors.

In another embodiment, a device for base calling is provided. The devicecomprises a biosensor. The biosensor has a sample surface. The samplesurface includes pixel areas and an array of wells overlying the pixelareas, including two wells per pixel area. The two wells per pixel areainclude a dominant well and a subordinate well. The dominant well has alarger cross section over the pixel area than the subordinate well.

The two wells can have different offsets relative to a center of thepixel area. During a sampling event, the pixel area can receivedifferent amounts of illumination from the two wells. Each of the twowells can hold at least one cluster during the sampling event. Duringthe sampling event, the pixel area can receive an amount of illuminationfrom a bright cluster in the dominant well that is greater than anamount of illumination received from a dim cluster in the subordinatewell.

The biosensor can be coupled to a signal processor. The signal processorcan be configured to receive and to process the plurality of sequencesof pixel signals to identify nucleotide bases present in a number N+M ofclusters from the number N of active sensors. For the bright and dimcluster, this can include mapping into at least four bins a first pixelsignal generated by a sensor corresponding to the pixel area during afirst illumination stage of the sampling event, mapping into at leastfour bins a second pixel signal generated by the sensor during a secondillumination stage of the sampling event, and logically combining themapping of the first and second pixel signals to identify the nucleotidebases present in the bright cluster and the dim cluster.

The biosensor also has an array of sensors configured to generate aplurality of sequences of pixel signals. The array has a number N ofactive sensors. The sensors in the array are disposed relative to thesample surface to generate respective pixel signals during the sequenceof sampling events from the number N of corresponding pixel areas of thesample surface to produce the plurality of sequences of pixel signals.The biosensor also has a communication port which outputs the pluralityof sequences of pixel signals.

In yet another embodiment, a device for base calling is provided. Thedevice comprises a biosensor. The biosensor has a sample surface. Thesample surface includes pixel areas and an array of wells overlying thepixel areas. The device further comprises an illumination system. Theillumination system illuminates the pixel areas with different angles ofillumination during a sequence of sampling events, including for asampling event in the sequence of sampling events illuminating each ofthe wells with off-axis illumination to produce asymmetricallyilluminated well regions in each of the wells.

The asymmetrically illuminated regions of a well can include at least adominant well region and a subordinate well region, such that during thesampling event the dominant well region is illuminated more than thesubordinate well region. The well can hold more than one cluster duringthe sampling event, with the dominant and subordinate well regions eachincluding a cluster. During the sampling event, a pixel area overlyingthe well can receive an amount of illumination from a bright cluster inthe dominant well region that is greater than an amount of illuminationreceived from a dim cluster in the subordinate well region.

The off-axis illumination can be at a forty-five degree angle. In someembodiments, one well overlies per pixel area. In other embodiments, twowells overlie per pixel area.

The biosensor can be coupled to a signal processor. The signal processorcan be configured to receive and to process the plurality of sequencesof pixel signals to identify nucleotide bases present in a number N+M ofclusters from the number N of active sensors. For the bright and dimcluster, this can include mapping into at least four bins a first pixelsignal generated by a sensor corresponding to the pixel area during afirst illumination stage of the sampling event, mapping into at leastfour bins a second pixel signal generated by the sensor during a secondillumination stage of the sampling event, and logically combining themapping of the first and second pixel signals to identify the nucleotidebases present in the bright cluster and the dim cluster.

The biosensor also has an array of sensors configured to generate aplurality of sequences of pixel signals. The array has a number N ofactive sensors. The sensors in the array are disposed relative to thesample surface to generate respective pixel signals during the sequenceof sampling events from the number N of corresponding pixel areas of thesample surface to produce the plurality of sequences of pixel signals.The biosensor also has a communication port which outputs the pluralityof sequences of pixel signals.

In accordance with another embodiment, a device for base calling isprovided that comprises a receptacle configured to hold a biosensor. Thebiosensor has (a) a sample surface that holds a plurality of clustersduring a sequence of sampling events, (b) an array of sensors configuredto generate a plurality of sequences of pixel signals, and (c) acommunication port which outputs the plurality of sequences of pixelsignals. Each sensor in the array senses information from one or moreclusters disposed in corresponding pixel areas of the sample surface togenerate a pixel signal in a sampling event. The array has a number N ofactive sensors and the sensors in the array are disposed relative to thesample surface to generate respective pixel signals during the sequenceof sampling events from the number N of corresponding pixel areas of thesample surface to produce the plurality of sequences of pixel signals.The device further comprises a signal processor coupled to thereceptacle. The signal processor is configured to receive and to processthe plurality of sequences of pixel signals to classify results of thesequence of sampling events on clusters in the plurality of clusters.The pixel signal for each sampling event in at least one sequence ofpixel signals in the plurality of sequences of pixel signals representssensed information from at least two clusters in the corresponding pixelarea. The signal processor uses the plurality of sequences of pixelsignals to classify results of the sequence of sampling events on anumber N+M of clusters in the plurality of clusters from the number N ofactive sensors, where Mis a positive integer.

The results of the sequence of sampling events can correspond tonucleotide bases in the clusters.

The sampling events can comprise two illumination stages in timesequence, and said at least one sequence of pixel signals in theplurality of sequences of pixel signals can include one pixel signalincluding information from at least two clusters in the correspondingpixel area from each of the two illumination stages.

The signal processor can include logic to classify results for twoclusters from the sequences of pixel signals from said at least onesequence of pixel signals. The logic to classify results for twoclusters can include mapping a first pixel signal in said at least onesequence of pixel signals from a particular sensor into at least fourbins, and mapping a second pixel signal in said at least one sequence ofpixel signals into at least four bins, and logically combining themapping of the first and second pixel signals to classify the resultsfor two clusters.

The sensors in the array of sensors can comprise light detectors.

The sampling events can comprise two illumination stages in timesequence, and sequences of pixel signals in the plurality of sequencesof pixel signals include at least one pixel signal from each of the twoillumination stages. The first illumination stage can induceillumination from one or more clusters in the pixel areas of the sensorsindicating nucleotide bases A and T and the second illumination stageinduces illumination from one or more clusters in the pixel areas of thesensors indicating nucleotide bases C and T, and said classifyingresults comprises calling one of the nucleotide bases A, C, Tor G for atleast two clusters using said at least one sequence.

Clusters can be distributed unevenly over the pixel areas of the samplesurface, and the signal processor can execute time sequence and spatialanalysis of the plurality of sequences of pixel signals to detectpatterns of illumination corresponding to individual clusters on thesample surface, and to classify the results of the sampling events forthe individual clusters. The plurality of sequences of pixel signalsencodes differential crosstalk between at least two clusters resultingfrom their uneven distribution over the pixel areas.

The sample surface can comprise an array of wells overlying the pixelareas, including two wells per pixel area, the two wells per pixel areacan include a dominant well and a subordinate well, the dominant wellcan have a larger cross section over the pixel area than the subordinatewell.

The sample surface can comprise an array of wells overlying the pixelareas, and the sampling events can include at least one chemical stagewith a number K of illumination stages where K is a positive integer.The illumination stages of the K illumination stages can illuminate thepixel areas with different angles of illumination, and the sequences ofpixel signals can include the number K of pixel signals for the at leastone chemical stage of the sampling events.

The sample surface can comprise an array of wells overlying the pixelareas, and the sampling events can include a first chemical stage with anumber K of illumination stages where K is a positive integer. Theillumination stages of the K illumination stages can illuminate thepixel areas with different angles of illumination, and a second chemicalstage with a number J of illumination stages where J is a positiveinteger. The illumination stages of the K illumination stages in thefirst chemical stage and of the J illumination stages in the secondchemical stage can illuminate the wells in the array of wells withdifferent angles of illumination, and the sequences of pixel signals caninclude the number K of pixel signals for the first chemical stage plusthe number J of pixel signals for the second chemical stage of thesampling events.

In yet another embodiment, a biosensor for base calling is provided. Thebiosensor comprises a sampling device. The sampling device includes asample surface that has an array of pixel areas and a solid-state imagerthat has an array of sensors. Each sensor generates pixel signals ineach base calling cycle. Each pixel signal represents light gathered inone base calling cycle from one or more clusters in a correspondingpixel area of the sample surface. The biosensor further comprises asignal processor configured for connection to the sampling device. Thesignal processor receives and processes the pixel signals from thesensors for base calling in a base calling cycle, and uses the pixelsignals from fewer sensors than a number of clusters base called in thebase calling cycle. The pixel signals from the fewer sensors include atleast one pixel signal representing light gathered from at least twoclusters in the corresponding pixel area.

A pixel area can receive light from a well on the sample surface and thewell can hold more than one cluster during the base calling cycle.

A cluster can comprise a plurality of single-stranded fragments thathave an identical base sequence.

In a further embodiment, a method of base calling is provided. For abase calling cycle of a sequencing by synthesis (abbreviated SBS) run,the method includes detecting: (1) a first pixel signal that representslight gathered from at least two clusters in a first pixel area during afirst illumination stage of the base calling cycle and (2) a secondpixel signal that represents light gathered from said at least twoclusters in the first pixel area during a second illumination stage ofthe base calling cycle. The first pixel area underlies a plurality ofclusters that shares the first pixel area. The method includes using acombination of the first and second pixel signals to identify nucleotidebases incorporated onto each cluster of the at least two clusters duringthe base calling cycle.

The method can also include mapping the first pixel signal into at leastfour bins and mapping the second pixel signal into at least four bins,and combining the mapping of the first and second pixel signals toidentify the incorporated nucleotide bases.

The method can also include applying the method to identify thenucleotide bases incorporated onto the plurality of clusters at aplurality of pixel areas during the base calling cycle.

The method can also include repeating the method over successive basecalling cycles to identify the nucleotide bases incorporated onto theplurality of clusters at the plurality of pixel areas during each of thebase calling cycles.

For each of the base calling cycles, the method can also includedetecting and storing the first and second pixel signals emitted by theplurality of clusters at the plurality of pixel areas, and after thebase calling cycles, using the combination of the first and second pixelsignals to identify the nucleotide bases incorporated onto the pluralityof clusters at the plurality of pixel areas during each of the previousbase calling cycles.

The first pixel area can receive light from an associated well on asample surface. The first pixel area can receive light from more thanone associated well on the sample surface. The first and second pixelsignals can be gathered by a first sensor from the first pixel area. Thefirst and second pixel signals can be detected by a signal processorconfigured for processing pixel signals gathered by the first sensor.The first illumination stage can induce illumination from the first andsecond clusters to produce emissions from labeled nucleotide bases A andT and the second illumination stage can induce illumination from thefirst and second clusters to produce emissions from labeled nucleotidebases C and T.

In another embodiment, a method of identifying pixel areas with morethan one cluster on a sample surface of a biosensor and base callingclusters at the identified pixel areas is provided. The method includesperforming a plurality of base calling cycles, each base calling cyclehaving a first illumination stage and a second illumination stage. Themethod includes capturing at a sensor associated with a pixel area ofthe sample surface, (1) a first set of intensity values generated duringthe first illumination stage of the base calling cycles, and (2) asecond set of intensity values generated during the second illuminationstage of the base calling cycles. The method includes fitting the firstand second sets of intensity values to a set of distributions using asignal processor and, based on the fitting, classifying the pixel areaas having more than one cluster. For a successive base calling cycle,the method includes detecting the first and second sets of intensityvalues for a cluster group at the pixel area using the signal processor,and selecting a distribution for the cluster group. The distributionidentifies a nucleotide base present in each cluster of the clustergroup.

The method can include fitting comprises using one or more algorithms,including a k-means clustering algorithm, a k-means-like clusteringalgorithm, an expectation maximization algorithm, and a histogram basedalgorithm.

The method can include normalizing the intensity values.

The pixel area can receive light from an associated well on the samplesurface.

In another embodiment, a device for base calling is provided. The devicecomprises a receptacle configured to hold a biosensor. The biosensor hasa sample surface. The sample surface includes pixel areas that underlaya plurality of clusters during a sequence of sampling events such thatthe clusters are distributed unevenly over the pixel areas. Thebiosensor also has an array of sensors configured to generate aplurality of sequences of pixel signals. Each sensor in the array sensesinformation from one or more clusters disposed in corresponding pixelareas of the sample surface to generate a pixel signal in a samplingevent. The array has a number N of active sensors. The sensors in thearray are disposed relative to the sample surface to generate respectivepixel signals during the sequence of sampling events from the number Nof corresponding pixel areas of the sample surface to produce theplurality of sequences of pixel signals. The biosensor also has acommunication port which outputs the plurality of sequences of pixelsignals. The device further comprises a signal processor coupled to thereceptacle. The signal processor is configured to execute time sequenceand spatial analysis of the plurality of sequences of pixel signals todetect patterns of illumination corresponding to a number N+M ofindividual clusters on the sample surface from the number N of activesensors, where M is a positive integer, and to classify the results ofthe sequence of sampling events for the number N+M of individualclusters. The pixel signal for each sampling event in at least onesequence of pixel signals in the plurality of sequences of pixel signalsrepresents sensed information from at least two clusters in thecorresponding pixel area and the plurality of sequences of pixel signalsencodes differential crosstalk between the at least two clustersresulting from their uneven distribution over the pixel areas.

The signal processor can use the detected patterns of illumination tolocate the number N+M of individual clusters on the sample surface fromthe number N of active sensors.

In another embodiment, a device for base calling is provided. The devicecomprises a biosensor. The biosensor has a sample surface. The samplesurface includes pixel areas and an array of wells overlying the pixelareas, the biosensor including two wells and two clusters per pixelarea. The two wells per pixel area include a dominant well and asubordinate well. The dominant well has a larger cross section over thepixel area than the subordinate well.

The biosensor also has an array of sensors configured to generate aplurality of sequences of pixel signals. Each sensor in the array sensesinformation from the two clusters disposed in corresponding pixel areasof the sample surface to generate a pixel signal in a sampling event.The array has a number N of active sensors. The sensors in the array aredisposed relative to the sample surface to generate respective pixelsignals during the sequence of sampling events from the number N ofcorresponding pixel areas of the sample surface to produce the pluralityof sequences of pixel signals. The biosensor also has a communicationport which outputs the plurality of sequences of pixel signals.

The two wells can have different offsets relative to a center of thepixel area. During a sampling event, the pixel area can receivedifferent amounts of illumination from the two wells. The pixel signalfor each sampling event in at least one sequence of pixel signals in theplurality of sequences of pixel signals represents sensed informationfrom the two clusters in the corresponding pixel area. Each of the twowells can hold at least one cluster during the sampling event. Duringthe sampling event, the pixel area can receive an amount of illuminationfrom a bright cluster in the dominant well that is greater than anamount of illumination received from a dim cluster in the subordinatewell.

The biosensor can be coupled to a signal processor. The signal processorcan be configured to receive and to process the plurality of sequencesof pixel signals to identify nucleotide bases present in a number N+M ofclusters from the number N of active sensors. For the bright and dimcluster, this can include mapping into at least four bins a first pixelsignal generated by a sensor corresponding to the pixel area during afirst illumination stage of the sampling event, mapping into at leastfour bins a second pixel signal generated by the sensor during a secondillumination stage of the sampling event, and logically combining themapping of the first and second pixel signals to identify the nucleotidebases present in the bright cluster and the dim cluster.

In yet another embodiment, a device for base calling is provided. Thedevice comprises a biosensor. The biosensor has a sample surface. Thesample surface includes pixel areas and an array of wells overlying thepixel areas, with at least two clusters per pixel area. The devicefurther comprises an illumination system. The illumination systemilluminates the pixel areas with different angles of illumination duringa sequence of sampling events, including for a sampling event in thesequence of sampling events illuminating each of the wells with off-axisillumination to produce asymmetrically illuminated well regions in eachof the wells.

The asymmetrically illuminated regions of a well can include at least adominant well region and a subordinate well region, such that during thesampling event the dominant well region is illuminated more than thesubordinate well region. The well can hold more than one cluster duringthe sampling event, with the dominant and subordinate well regions eachincluding a cluster. During the sampling event, a pixel area overlyingthe well can receive an amount of illumination from a bright cluster inthe dominant well region that is greater than an amount of illuminationreceived from a dim cluster in the subordinate well region.

The off-axis illumination can be at a forty-five degree angle. In someembodiments, one well overlies per pixel area. In other embodiments, twowells overlie per pixel area.

The biosensor also has an array of sensors configured to generate aplurality of sequences of pixel signals. Each sensor in the array sensesinformation from the at least two clusters disposed in correspondingpixel areas of the sample surface to generate a pixel signal in asampling event. The array has a number N of active sensors. The sensorsin the array are disposed relative to the sample surface to generaterespective pixel signals during the sequence of sampling events from thenumber N of corresponding pixel areas of the sample surface to producethe plurality of sequences of pixel signals. The biosensor also has acommunication port which outputs the plurality of sequences of pixelsignals.

The biosensor can be coupled to a signal processor. The signal processorcan be configured to receive and to process the plurality of sequencesof pixel signals to identify nucleotide bases present in a number N+M ofclusters from the number N of active sensors. For the bright and dimcluster, this can include mapping into at least four bins a first pixelsignal generated by a sensor corresponding to the pixel area during afirst illumination stage of the sampling event, mapping into at leastfour bins a second pixel signal generated by the sensor during a secondillumination stage of the sampling event, and logically combining themapping of the first and second pixel signals to identify the nucleotidebases present in the bright cluster and the dim cluster.

Other features and aspects of the technology disclosed will becomeapparent from the following detailed description, taken in conjunctionwith the accompanying drawings, which illustrate, by way of example, thefeatures in accordance with embodiments of the technology disclosed.This brief description is not intended to limit the scope of anyinventions described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The color drawings also may be available in PAIRvia the Supplemental Content tab.

The present disclosure, in accordance with one or more embodiments, isdescribed in detail with reference to the following figures. The figuresare provided for purposes of illustration only and merely depict exampleembodiments. Furthermore, it should be noted that for clarity and easeof illustration, the elements in the figures have not necessarily beendrawn to scale.

Some of the figures included herein illustrate various embodiments ofthe technology disclosed from different viewing angles. Although theaccompanying descriptive text may refer to such views as “top,” “bottom”or “side” views, such references are merely descriptive and do not implyor require that the technology disclosed be implemented or used in aparticular spatial orientation unless explicitly stated otherwise.

FIG. 1 is a block diagram of a base calling system in accordance withone embodiment.

FIG. 2 is a block diagram of a system controller that can be used in thesystem of FIG. 1 .

FIG. 3 illustrates a cross-section of a biosensor that can be used invarious embodiments. FIG. 3 's biosensor has pixel areas that can eachhold more than one cluster during a base calling cycle (e.g., 2 clustersper pixel area).

FIG. 4 shows a cross-section of a biosensor that can be used in variousembodiments. FIG. 4 's biosensor has wells that can each hold more thanone cluster during a base calling cycle (e.g., 2 clusters per well).

FIGS. 5A and 5B are scatter plots that depict base calling of bright anddim clusters of a cluster pair using their respective pixel signalsdetected by a shared sensor (or pixel) in accordance with oneembodiment.

FIG. 6 is a scatter plot that depicts sixteen distributions produced byintensity values from bright and dim clusters of a cluster pair inaccordance with one embodiment.

FIG. 7A is a detection table that illustrates a base calling scheme forone dye and two illumination stage sequencing protocol in accordancewith one embodiment.

FIG. 7B is a base calling table that shows a classification scheme forclassifying combined pixel signals from bright and dim clusters of acluster pair into one of sixteen bins in accordance with one embodiment.

FIG. 8 shows a method of base calling by analyzing pixel signals emittedby a plurality of clusters that share a pixel area in accordance withone embodiment.

FIG. 9 depicts a method of identifying pixel areas with more than onecluster on a sample surface of a biosensor and base calling clusters atthe identified pixel areas in accordance with one embodiment.

FIG. 10 illustrates a top plan view of a sample surface having pixelareas on which a plurality of clusters is unevenly distributed inaccordance with one embodiment.

FIG. 11A illustrates a side view of a sample surface having two wellsper pixel area including a dominant well and a subordinate well inaccordance with one embodiment.

FIG. 11B depicts a top plan view of the sample surface of FIG. 11A.

FIGS. 12A and 12B show off-axis illumination of a well overlying a pixelarea of a sample surface.

FIG. 12C illustrates asymmetrically illuminated well regions produced bythe off-axis illumination of FIGS. 12A and 12B in accordance with oneembodiment.

DETAILED DESCRIPTION

Embodiments described herein may be used in various biological orchemical processes and systems for academic or commercial analysis. Morespecifically, embodiments described herein may be used in variousprocesses and systems where it is desired to detect an event, property,quality, or characteristic that is indicative of a desired reaction. Forexample, embodiments described herein include cartridges, biosensors,and their components as well as bioassay systems that operate withcartridges and biosensors. In particular embodiments, the cartridges andbiosensors include a flow cell and one or more sensors, pixels, lightdetectors, or photodiodes that are coupled together in a substantiallyunitary structure.

The bioassay systems may be configured to perform a plurality of desiredreactions that may be detected individually or collectively. Thebiosensors and bioassay systems may be configured to perform numerouscycles in which the plurality of desired reactions occurs in parallel.For example, the bioassay systems may be used to sequence a dense arrayof DNA features through iterative cycles of enzymatic manipulation anddata acquisition. As such, the cartridges and biosensors may include oneor more microfluidic channels that deliver reagents or other reactioncomponents to a reaction site. In some embodiments, the reaction sitesare unevenly distributed across a substantially planar surface. In otherembodiments, the reaction sites are patterned across a substantiallyplanar surface in a predetermined manner. Each of the reaction sites maybe associated with one or more sensors, pixels, light detectors, orphotodiodes that detect light from the associated reaction site. Yet inother embodiments, the reaction sites are located in reaction chambers(or wells) that compartmentalize the desired reactions therein.

The following detailed description of certain embodiments will be betterunderstood when read in conjunction with the appended drawings. To theextent that the figures illustrate diagrams of the functional blocks ofvarious embodiments, the functional blocks are not necessarilyindicative of the division between hardware circuitry. Thus, forexample, one or more of the functional blocks (e.g., processors ormemories) may be implemented in a single piece of hardware (e.g., ageneral purpose signal processor or random access memory, hard disk, orthe like). Similarly, the programs may be standalone programs, may beincorporated as subroutines in an operating system, may be functions inan installed software package, and the like. It should be understoodthat the various embodiments are not limited to the arrangements andinstrumentality shown in the drawings.

As used herein, an element or step recited in the singular and proceededwith the word “a” or “an” should be understood as not excluding pluralof said elements or steps, unless such exclusion is explicitly stated.Furthermore, references to “one embodiment” are not intended to beinterpreted as excluding the existence of additional embodiments thatalso incorporate the recited features. Moreover, unless explicitlystated to the contrary, embodiments “comprising” or “having” or“including” an element or a plurality of elements having a particularproperty may include additional elements whether or not they have thatproperty.

As used herein, a “desired reaction” includes a change in at least oneof a chemical, electrical, physical, or optical property (or quality) ofan analyte-of-interest. In particular embodiments, the desired reactionis a positive binding event (e.g., incorporation of a fluorescentlylabeled biomolecule with the analyte-of-interest). More generally, thedesired reaction may be a chemical transformation, chemical change, orchemical interaction. The desired reaction may also be a change inelectrical properties. For example, the desired reaction may be a changein ion concentration within a solution. Exemplary reactions include, butare not limited to, chemical reactions such as reduction, oxidation,addition, elimination, rearrangement, esterification, amidation,etherifi cation, cyclization, or substitution; binding interactions inwhich a first chemical binds to a second chemical; dissociationreactions in which two or more chemicals detach from each other;fluorescence; luminescence; bioluminescence; chemiluminescence; andbiological reactions, such as nucleic acid replication, nucleic acidamplification, nucleic acid hybridization, nucleic acid ligation,phosphorylation, enzymatic catalysis, receptor binding, or ligandbinding. The desired reaction can also be an addition or elimination ofa proton, for example, detectable as a change in pH of a surroundingsolution or environment. An additional desired reaction can be detectingthe flow of ions across a membrane (e.g., natural or synthetic bilayermembrane), for example as ions flow through a membrane the current isdisrupted and the disruption can be detected.

In particular embodiments, the desired reaction includes theincorporation of a fluorescently-labeled molecule to an analyte. Theanalyte may be an oligonucleotide and the fluorescently-labeled moleculemay be a nucleotide. The desired reaction may be detected when anexcitation light is directed toward the oligonucleotide having thelabeled nucleotide, and the fluorophore emits a detectable fluorescentsignal. In alternative embodiments, the detected fluorescence is aresult of chemiluminescence or bioluminescence. A desired reaction mayalso increase fluorescence (or Forster) resonance energy transfer(FRET), for example, by bringing a donor fluorophore in proximity to anacceptor fluorophore, decrease FRET by separating donor and acceptorfluorophores, increase fluorescence by separating a quencher from afluorophore or decrease fluorescence by co-locating a quencher andfluorophore.

As used herein, a “reaction component” or “reactant” includes anysubstance that may be used to obtain a desired reaction. For example,reaction components include reagents, enzymes, samples, otherbiomolecules, and buffer solutions. The reaction components aretypically delivered to a reaction site in a solution and/or immobilizedat a reaction site. The reaction components may interact directly orindirectly with another substance, such as the analyte-of-interest.

As used herein, the term “reaction site” is a localized region where adesired reaction may occur. A reaction site may include support surfacesof a substrate where a substance may be immobilized thereon. Forexample, a reaction site may include a substantially planar surface in achannel of a flow cell that has a colony of nucleic acids thereon.Typically, but not always, the nucleic acids in the colony have the samesequence, being for example, clonal copies of a single stranded ordouble stranded template. However, in some embodiments a reaction sitemay contain only a single nucleic acid molecule, for example, in asingle stranded or double stranded form. Furthermore, a plurality ofreaction sites may be unevenly distributed along the support surface orarranged in a predetermined manner (e.g., side-by-side in a matrix, suchas in microarrays). A reaction site can also include a reaction chamber(or well) that at least partially defines a spatial region or volumeconfigured to compartmentalize the desired reaction.

This application uses the terms “reaction chamber” and “well”interchangeably. As used herein, the term “reaction chamber” or “well”includes a spatial region that is in fluid communication with a flowchannel. The reaction chamber may be at least partially separated fromthe surrounding environment or other spatial regions. For example, aplurality of reaction chambers may be separated from each other byshared walls. As a more specific example, the reaction chamber mayinclude a cavity defined by interior surfaces of a well and have anopening or aperture so that the cavity may be in fluid communicationwith a flow channel. Biosensors including such reaction chambers aredescribed in greater detail in international application no.PCT/US2011/057111, filed on Oct. 20, 2011, which is incorporated hereinby reference in its entirety.

In some embodiments, the reaction chambers are sized and shaped relativeto solids (including semi-solids) so that the solids may be inserted,fully or partially, therein. For example, the reaction chamber may besized and shaped to accommodate only one capture bead. The capture beadmay have clonally amplified DNA or other substances thereon.Alternatively, the reaction chamber may be sized and shaped to receivean approximate number of beads or solid substrates. As another example,the reaction chambers may also be filled with a porous gel or substancethat is configured to control diffusion or filter fluids that may flowinto the reaction chamber.

In some embodiments, sensors (e.g., light detectors, photodiodes) areassociated with corresponding pixel areas of a sample surface of abiosensor. As such, a pixel area is a geometrical construct thatrepresents an area on the biosensor' s sample surface for one sensor (orpixel). A sensor that is associated with a pixel area detects lightemissions gathered from the associated pixel area when a desiredreaction has occurred at a reaction site or a reaction chamber overlyingthe associated pixel area. In a flat surface embodiment, the pixel areascan overlap. In some cases, a plurality of sensors may be associatedwith a single reaction site or a single reaction chamber. In othercases, a single sensor may be associated with a group of reaction sitesor a group of reaction chambers.

As used herein, a “biosensor” includes a structure having a plurality ofreaction sites and/or reaction chambers (or wells). A biosensor mayinclude a solid-state imaging device (e.g., CCD or CMOS imager) and,optionally, a flow cell mounted thereto. The flow cell may include atleast one flow channel that is in fluid communication with the reactionsites and/or the reaction chambers. As one specific example, thebiosensor is configured to fluidically and electrically couple to abioassay system. The bioassay system may deliver reactants to thereaction sites and/or the reaction chambers according to a predeterminedprotocol (e.g., sequencing-by-synthesis) and perform a plurality ofimaging events. For example, the bioassay system may direct solutions toflow along the reaction sites and/or the reaction chambers. At least oneof the solutions may include four types of nucleotides having the sameor different fluorescent labels. The nucleotides may bind tocorresponding oligonucleotides located at the reaction sites and/or thereaction chambers. The bioassay system may then illuminate the reactionsites and/or the reaction chambers using an excitation light source(e.g., solid-state light sources, such as light-emitting diodes orLEDs). The excitation light may have a predetermined wavelength orwavelengths, including a range of wavelengths. The excited fluorescentlabels provide emission signals that may be captured by the sensors.

In alternative embodiments, the biosensor may include electrodes orother types of sensors configured to detect other identifiableproperties. For example, the sensors may be configured to detect achange in ion concentration. In another example, the sensors may beconfigured to detect the ion current flow across a membrane.

As used herein, a “cartridge” includes a structure that is configured tohold a biosensor. In some embodiments, the cartridge may includeadditional features, such as the light source (e.g., LEDs) that areconfigured to provide excitation light to the reaction sites and/or thereaction chambers of the biosensor. The cartridge may also include afluidic storage system (e.g., storage for reagents, sample, and buffer)and a fluidic control system (e.g., pumps, valves, and the like) forfluidically transporting reaction components, sample, and the like tothe reaction sites and/or the reaction chambers. For example, after thebiosensor is prepared or manufactured, the biosensor may be coupled to ahousing or container of the cartridge. In some embodiments, thebiosensors and the cartridges may be self-contained, disposable units.However, other embodiments may include an assembly with removable partsthat allow a user to access an interior of the biosensor or cartridgefor maintenance or replacement of components or samples. The biosensorand the cartridge may be removably coupled or engaged to larger bioassaysystems, such as a sequencing system, that conducts controlled reactionstherein.

As used herein, when the terms “removably” and “coupled” (or “engaged”)are used together to describe a relationship between the biosensor (orcartridge) and a system receptacle or interface of a bioassay system,the term is intended to mean that a connection between the biosensor (orcartridge) and the system receptacle is readily separable withoutdestroying or damaging the system receptacle and/or the biosensor (orcartridge). Components are readily separable when the components may beseparated from each other without undue effort or a significant amountof time spent in separating the components. For example, the biosensor(or cartridge) may be removably coupled or engaged to the systemreceptacle in an electrical manner such that the mating contacts of thebioassay system are not destroyed or damaged. The biosensor (orcartridge) may also be removably coupled or engaged to the systemreceptacle in a mechanical manner such that the features that hold thebiosensor (or cartridge) are not destroyed or damaged. The biosensor (orcartridge) may also be removably coupled or engaged to the systemreceptacle in a fluidic manner such that the ports of the systemreceptacle are not destroyed or damaged. The system receptacle or acomponent is not considered to be destroyed or damaged if, for example,only a simple adjustment to the component (e.g., realignment) or asimple replacement (e.g., replacing a nozzle) is required.

As used herein, a “cluster” is a colony of similar or identicalmolecules or nucleotide sequences or DNA strands. For example, a clustercan be an amplified oligonucleotide or any other group of apolynucleotide or polypeptide with a same or similar sequence. In otherembodiments, a cluster can be any element or group of elements thatoccupy a physical area on a sample surface. In embodiments, clusters areimmobilized to a reaction site and/or a reaction chamber during a basecalling cycle.

As used herein, the term “immobilized,” when used with respect to abiomolecule or biological or chemical substance, includes substantiallyattaching the biomolecule or biological or chemical substance at amolecular level to a surface. For example, a biomolecule or biologicalor chemical substance may be immobilized to a surface of the substratematerial using adsorption techniques including non-covalent interactions(e.g., electrostatic forces, van der Waals, and dehydration ofhydrophobic interfaces) and covalent binding techniques where functionalgroups or linkers facilitate attaching the biomolecules to the surface.Immobilizing biomolecules or biological or chemical substances to asurface of a substrate material may be based upon the properties of thesubstrate surface, the liquid medium carrying the biomolecule orbiological or chemical substance, and the properties of the biomoleculesor biological or chemical substances themselves. In some cases, asubstrate surface may be functionalized (e.g., chemically or physicallymodified) to facilitate immobilizing the biomolecules (or biological orchemical substances) to the substrate surface. The substrate surface maybe first modified to have functional groups bound to the surface. Thefunctional groups may then bind to biomolecules or biological orchemical substances to immobilize them thereon. A substance can beimmobilized to a surface via a gel, for example, as described in USPatent Publ. No. US 2011/0059865 A1, which is incorporated herein byreference.

In some embodiments, nucleic acids can be attached to a surface andamplified using bridge amplification. Useful bridge amplificationmethods are described, for example, in U.S. Pat. No. 5,641,658; WO2007/010251, U.S. Pat. No. 6,090,592; U.S. Patent Publ. No. 2002/0055100A1; U.S. Pat. No. 7,115,400; U.S. Patent Publ. No. 2004/0096853 A1; U.S.Patent Publ. No. 2004/0002090 A1; U.S. Patent Publ. No. 2007/0128624 A1;and U.S. Patent Publ. No. 2008/0009420 A1, each of which is incorporatedherein in its entirety. Another useful method for amplifying nucleicacids on a surface is rolling circle amplification (RCA), for example,using methods set forth in further detail below. In some embodiments,the nucleic acids can be attached to a surface and amplified using oneor more primer pairs. For example, one of the primers can be in solutionand the other primer can be immobilized on the surface (e.g.,5′-attached). By way of example, a nucleic acid molecule can hybridizeto one of the primers on the surface followed by extension of theimmobilized primer to produce a first copy of the nucleic acid. Theprimer in solution then hybridizes to the first copy of the nucleic acidwhich can be extended using the first copy of the nucleic acid as atemplate. Optionally, after the first copy of the nucleic acid isproduced, the original nucleic acid molecule can hybridize to a secondimmobilized primer on the surface and can be extended at the same timeor after the primer in solution is extended. In any embodiment, repeatedrounds of extension (e.g., amplification) using the immobilized primerand primer in solution provide multiple copies of the nucleic acid.

In particular embodiments, the assay protocols executed by the systemsand methods described herein include the use of natural nucleotides andalso enzymes that are configured to interact with the naturalnucleotides. Natural nucleotides include, for example, ribonucleotides(RNA) or deoxyribonucleotides (DNA). Natural nucleotides can be in themono-, di-, or tri-phosphate form and can have a base selected fromadenine (A), thymine (T), uracil (U), guanine (G) or cytosine (C). Itwill be understood however that non-natural nucleotides, modifiednucleotides or analogs of the aforementioned nucleotides can be used.Some examples of useful non-natural nucleotides are set forth below inregard to reversible terminator-based sequencing by synthesis methods.

In embodiments that include reaction chambers, items or solid substances(including semi-solid substances) may be disposed within the reactionchambers. When disposed, the item or solid may be physically held orimmobilized within the reaction chamber through an interference fit,adhesion, or entrapment. Exemplary items or solids that may be disposedwithin the reaction chambers include polymer beads, pellets, agarosegel, powders, quantum dots, or other solids that may be compressedand/or held within the reaction chamber. In particular embodiments, anucleic acid superstructure, such as a DNA ball, can be disposed in orat a reaction chamber, for example, by attachment to an interior surfaceof the reaction chamber or by residence in a liquid within the reactionchamber. A DNA ball or other nucleic acid superstructure can bepreformed and then disposed in or at the reaction chamber.Alternatively, a DNA ball can be synthesized in situ at the reactionchamber. A DNA ball can be synthesized by rolling circle amplificationto produce a concatamer of a particular nucleic acid sequence and theconcatamer can be treated with conditions that form a relatively compactball. DNA balls and methods for their synthesis are described, forexample in, U.S. Patent Publication Nos. 2008/0242560 A1 or 2008/0234136A1, each of which is incorporated herein in its entirety. A substancethat is held or disposed in a reaction chamber can be in a solid,liquid, or gaseous state.

As used herein, “base calling” identifies a nucleotide base in a nucleicacid sequence. Base calling refers to the process of determining a basecall (A, C, G, T) for every cluster at a specific cycle. As an example,base calling can be performed utilizing four-channel, two-channel orone-channel methods and systems described in the incorporated materialsof U.S. Patent Application Publication No. 2013/0079232. In particularembodiments, a base calling cycle is referred to as a “sampling event.”In one dye and two-channel sequencing protocol, a sampling eventcomprises two illumination stages in time sequence, such that a pixelsignal is generated at each stage. The first illumination stage inducesillumination from a given cluster indicating nucleotide bases A and Tina AT pixel signal, and the second illumination stage inducesillumination from a given cluster indicating nucleotide bases C and T ina CT pixel signal.

Base Calling System

FIG. 1 is a block diagram of a base calling system 100 in accordancewith one embodiment. The base calling system 100 may operate to obtainany information or data that relates to at least one of a biological orchemical substance. In some embodiments, the base calling system 100 isa workstation that may be similar to a bench-top device or desktopcomputer. For example, a majority (or all) of the systems and componentsfor conducting the desired reactions can be within a common housing 116.

In particular embodiments, the base calling system 100 is a nucleic acidsequencing system (or sequencer) configured for various applications,including but not limited to de novo sequencing, resequencing of wholegenomes or target genomic regions, and metagenomics. The sequencer mayalso be used for DNA or RNA analysis. In some embodiments, the basecalling system 100 may also be configured to generate reaction sites ina biosensor. For example, the base calling system 100 may be configuredto receive a sample and generate surface attached clusters of clonallyamplified nucleic acids derived from the sample. Each cluster mayconstitute or be part of a reaction site in the biosensor.

The exemplary base calling system 100 may include a system receptacle orinterface 112 that is configured to interact with a biosensor 102 toperform desired reactions within the biosensor 102. In the followingdescription with respect to FIG. 1 , the biosensor 102 is loaded intothe system receptacle 112. However, it is understood that a cartridgethat includes the biosensor 102 may be inserted into the systemreceptacle 112 and in some states the cartridge can be removedtemporarily or permanently. As described above, the cartridge mayinclude, among other things, fluidic control and fluidic storagecomponents.

In particular embodiments, the base calling system 100 is configured toperform a large number of parallel reactions within the biosensor 102.The biosensor 102 includes one or more reaction sites where desiredreactions can occur. The reaction sites may be, for example, immobilizedto a solid surface of the biosensor or immobilized to beads (or othermovable substrates) that are located within corresponding reactionchambers of the biosensor. The reaction sites can include, for example,clusters of clonally amplified nucleic acids. The biosensor 102 mayinclude a solid-state imaging device (e.g., CCD or CMOS imager) and aflow cell mounted thereto. The flow cell may include one or more flowchannels that receive a solution from the base calling system 100 anddirect the solution toward the reaction sites. Optionally, the biosensor102 can be configured to engage a thermal element for transferringthermal energy into or out of the flow channel.

The base calling system 100 may include various components, assemblies,and systems (or sub-systems) that interact with each other to perform apredetermined method or assay protocol for biological or chemicalanalysis. For example, the base calling system 100 includes a systemcontroller 104 that may communicate with the various components,assemblies, and sub-systems of the base calling system 100 and also thebiosensor 102. For example, in addition to the system receptacle 112,the base calling system 100 may also include a fluidic control system106 to control the flow of fluid throughout a fluid network of the basecalling system 100 and the biosensor 102; a fluid storage system 108that is configured to hold all fluids (e.g., gas or liquids) that may beused by the bioassay system; a temperature control system 110 that mayregulate the temperature of the fluid in the fluid network, the fluidstorage system 108, and/or the biosensor 102; and an illumination system109 that is configured to illuminate the biosensor 102. As describedabove, if a cartridge having the biosensor 102 is loaded into the systemreceptacle 112, the cartridge may also include fluidic control andfluidic storage components.

Also shown, the base calling system 100 may include a user interface 114that interacts with the user. For example, the user interface 114 mayinclude a display 113 to display or request information from a user anda user input device 115 to receive user inputs. In some embodiments, thedisplay 113 and the user input device 115 are the same device. Forexample, the user interface 114 may include a touch-sensitive displayconfigured to detect the presence of an individual's touch and alsoidentify a location of the touch on the display. However, other userinput devices 115 may be used, such as a mouse, touchpad, keyboard,keypad, handheld scanner, voice-recognition system, motion-recognitionsystem, and the like. As will be discussed in greater detail below, thebase calling system 100 may communicate with various components,including the biosensor 102 (e.g., in the form of a cartridge), toperform the desired reactions. The base calling system 100 may also beconfigured to analyze data obtained from the biosensor to provide a userwith desired information.

The system controller 104 may include any processor-based ormicroprocessor-based system, including systems using microcontrollers,reduced instruction set computers (RISC), application specificintegrated circuits (ASICs), field programmable gate array (FPGAs),logic circuits, and any other circuit or processor capable of executingfunctions described herein. The above examples are exemplary only, andare thus not intended to limit in any way the definition and/or meaningof the term system controller. In the exemplary embodiment, the systemcontroller 104 executes a set of instructions that are stored in one ormore storage elements, memories, or modules in order to at least one ofobtain and analyze detection data. Detection data can include aplurality of sequences of pixel signals, such that a sequence of pixelsignals from each of the millions of sensors (or pixels) can be detectedover many base calling cycles. Storage elements may be in the form ofinformation sources or physical memory elements within the base callingsystem 100.

The set of instructions may include various commands that instruct thebase calling system 100 or biosensor 102 to perform specific operationssuch as the methods and processes of the various embodiments describedherein. The set of instructions may be in the form of a softwareprogram, which may form part of a tangible, non-transitory computerreadable medium or media. As used herein, the terms “software” and“firmware” are interchangeable, and include any computer program storedin memory for execution by a computer, including RAM memory, ROM memory,EPROM memory, EEPROM memory, and non-volatile RAM (NVRAM) memory. Theabove memory types are exemplary only, and are thus not limiting as tothe types of memory usable for storage of a computer program.

The software may be in various forms such as system software orapplication software. Further, the software may be in the form of acollection of separate programs, or a program module within a largerprogram or a portion of a program module. The software also may includemodular programming in the form of object-oriented programming. Afterobtaining the detection data, the detection data may be automaticallyprocessed by the base calling system 100, processed in response to userinputs, or processed in response to a request made by another processingmachine (e.g., a remote request through a communication link). In theillustrated embodiment, the system controller 104 includes the signalprocessor 138. In other embodiments, system controller 104 does notinclude the signal processor 138 and instead has access to the signalprocessor 138 (e.g., the signal processor 138 may be separately hostedon cloud).

The system controller 104 may be connected to the biosensor 102 and theother components of the base calling system 100 via communication links.The system controller 104 may also be communicatively connected tooff-site systems or servers. The communication links may be hardwired,corded, or wireless. The system controller 104 may receive user inputsor commands, from the user interface 114 and the user input device 115.

The fluidic control system 106 includes a fluid network and isconfigured to direct and regulate the flow of one or more fluids throughthe fluid network. The fluid network may be in fluid communication withthe biosensor 102 and the fluid storage system 108. For example, selectfluids may be drawn from the fluid storage system 108 and directed tothe biosensor 102 in a controlled manner, or the fluids may be drawnfrom the biosensor 102 and directed toward, for example, a wastereservoir in the fluid storage system 108. Although not shown, thefluidic control system 106 may include flow sensors that detect a flowrate or pressure of the fluids within the fluid network. The sensors maycommunicate with the system controller 104.

The temperature control system 110 is configured to regulate thetemperature of fluids at different regions of the fluid network, thefluid storage system 108, and/or the biosensor 102. For example, thetemperature control system 110 may include a thermocycler thatinterfaces with the biosensor 102 and controls the temperature of thefluid that flows along the reaction sites in the biosensor 102. Thetemperature control system 110 may also regulate the temperature ofsolid elements or components of the base calling system 100 or thebiosensor 102. Although not shown, the temperature control system 110may include sensors to detect the temperature of the fluid or othercomponents. The sensors may communicate with the system controller 104.

The fluid storage system 108 is in fluid communication with thebiosensor 102 and may store various reaction components or reactantsthat are used to conduct the desired reactions therein. The fluidstorage system 108 may also store fluids for washing or cleaning thefluid network and biosensor 102 and for diluting the reactants. Forexample, the fluid storage system 108 may include various reservoirs tostore samples, reagents, enzymes, other biomolecules, buffer solutions,aqueous, and non-polar solutions, and the like. Furthermore, the fluidstorage system 108 may also include waste reservoirs for receiving wasteproducts from the biosensor 102. In embodiments that include acartridge, the cartridge may include one or more of a fluid storagesystem, fluidic control system or temperature control system.Accordingly, one or more of the components set forth herein as relatingto those systems can be contained within a cartridge housing. Forexample, a cartridge can have various reservoirs to store samples,reagents, enzymes, other biomolecules, buffer solutions, aqueous, andnon-polar solutions, waste, and the like. As such, one or more of afluid storage system, fluidic control system or temperature controlsystem can be removably engaged with a bioassay system via a cartridgeor other biosensor.

The illumination system 109 may include alight source (e.g., one or moreLEDs) and a plurality of optical components to illuminate the biosensor.Examples of light sources may include lasers, arc lamps, LEDs, or laserdiodes. The optical components may be, for example, reflectors,dichroics, beam splitters, collimators, lenses, filters, wedges, prisms,mirrors, detectors, and the like. In embodiments that use anillumination system, the illumination system 109 may be configured todirect an excitation light to reaction sites. As one example,fluorophores may be excited by green wavelengths of light, as such thewavelength of the excitation light may be approximately 532 nm. In oneembodiment, the illumination system 109 is configured to produceillumination that is parallel to a surface normal of a surface of thebiosensor 102. In another embodiment, the illumination system 109 isconfigured to produce illumination that is off-angle relative to thesurface normal of the surface of the biosensor 102. In yet anotherembodiment, the illumination system 109 is configured to produceillumination that has plural angles, including some parallelillumination and some off-angle illumination.

The system receptacle or interface 112 is configured to engage thebiosensor 102 in at least one of a mechanical, electrical, and fluidicmanner. The system receptacle 112 may hold the biosensor 102 in adesired orientation to facilitate the flow of fluid through thebiosensor 102. The system receptacle 112 may also include electricalcontacts that are configured to engage the biosensor 102 so that thebase calling system 100 may communicate with the biosensor 102 and/orprovide power to the biosensor 102. Furthermore, the system receptacle112 may include fluidic ports (e.g., nozzles) that are configured toengage the biosensor 102. In some embodiments, the biosensor 102 isremovably coupled to the system receptacle 112 in a mechanical manner,in an electrical manner, and also in a fluidic manner.

In addition, the base calling system 100 may communicate remotely withother systems or networks or with other bioassay systems 100. Detectiondata obtained by the bioassay system(s) 100 may be stored in a remotedatabase.

FIG. 2 is a block diagram of a system controller 104 that can be used inthe system of FIG. 1 . In one embodiment, the system controller 104includes one or more processors or modules that can communicate with oneanother. Each of the processors or modules may include an algorithm(e.g., instructions stored on a tangible and/or non-transitory computerreadable storage medium) or sub-algorithms to perform particularprocesses. The system controller 104 is illustrated conceptually as acollection of modules, but may be implemented utilizing any combinationof dedicated hardware boards, DSPs, processors, etc. Alternatively, thesystem controller 104 may be implemented utilizing an off-the-shelf PCwith a single processor or multiple processors, with the functionaloperations distributed between the processors. As a further option, themodules described below may be implemented utilizing a hybridconfiguration in which certain modular functions are performed utilizingdedicated hardware, while the remaining modular functions are performedutilizing an off-the-shelf PC and the like. The modules also may beimplemented as software modules within a processing unit.

During operation, a communication port 120 may transmit information(e.g. commands) to or receive information (e.g., data) from thebiosensor 102 (FIG. 1 ) and/or the sub-systems 106, 108, 110 (FIG. 1 ).In embodiments, the communication port 120 may output a plurality ofsequences of pixel signals. A communication link 122 may receive userinput from the user interface 114 (FIG. 1 ) and transmit data orinformation to the user interface 114. Data from the biosensor 102 orsub-systems 106, 108, 110 may be processed by the system controller 104in real-time during a bioassay session. Additionally or alternatively,data may be stored temporarily in a system memory during a bioassaysession and processed in slower than real-time or off-line operation.

As shown in FIG. 2 , the system controller 104 may include a pluralityof modules 131-139 that communicate with a main control module 130. Themain control module 130 may communicate with the user interface 114(FIG. 1 ). Although the modules 131-139 are shown as communicatingdirectly with the main control module 130, the modules 131-139 may alsocommunicate directly with each other, the user interface 114, and thebiosensor 102. Also, the modules 131-139 may communicate with the maincontrol module 130 through the other modules.

The plurality of modules 131-139 include system modules 131-133, 139that communicate with the sub-systems 106, 108, 110, and 111,respectively. The fluidic control module 131 may communicate with thefluidic control system 106 to control the valves and flow sensors of thefluid network for controlling the flow of one or more fluids through thefluid network. The fluid storage module 132 may notify the user whenfluids are low or when the waste reservoir is at or near capacity. Thefluid storage module 132 may also communicate with the temperaturecontrol module 133 so that the fluids may be stored at a desiredtemperature. The illumination module 139 may communicate with theillumination system 109 to illuminate the reaction sites at designatedtimes during a protocol, such as after the desired reactions (e.g.,binding events) have occurred. In some embodiments, the illuminationmodule 139 may communicate with the illumination system 109 toilluminate the reaction sites at designated angles.

The plurality of modules 131-139 may also include a device module 134that communicates with the biosensor 102 and an identification module135 that determines identification information relating to the biosensor102. The device module 134 may, for example, communicate with the systemreceptacle 112 to confirm that the biosensor has established anelectrical and fluidic connection with the base calling system 100. Theidentification module 135 may receive signals that identify thebiosensor 102. The identification module 135 may use the identity of thebiosensor 102 to provide other information to the user. For example, theidentification module 135 may determine and then display a lot number, adate of manufacture, or a protocol that is recommended to be run withthe biosensor 102.

The plurality of modules 131-139 may also include a signal processingmodule or signal processor 138 that receives and analyzes the signaldata (e.g., image data) from the biosensor 102. Signal processor 138includes memory 140 (e.g., RAM or Flash) to store detection data.Detection data can include a plurality of sequences of pixel signals,such that a sequence of pixel signals from each of the millions ofsensors (or pixels) can be detected over many base calling cycles. Thesignal data may be stored for subsequent analysis or may be transmittedto the user interface 114 to display desired information to the user. Insome embodiments, the signal data may be processed by the solid-stateimager (e.g., CMOS image sensor) before the signal processor 138receives the signal data.

Protocol modules 136 and 137 communicate with the main control module130 to control the operation of the sub-systems 106, 108, and 110 whenconducting predetermined assay protocols. The protocol modules 136 and137 may include sets of instructions for instructing the base callingsystem 100 to perform specific operations pursuant to predeterminedprotocols. As shown, the protocol module may be asequencing-by-synthesis (SBS) module 136 that is configured to issuevarious commands for performing sequencing-by-synthesis processes. InSBS, extension of a nucleic acid primer along a nucleic acid template ismonitored to determine the sequence of nucleotides in the template. Theunderlying chemical process can be polymerization (e.g. as catalyzed bya polymerase enzyme) or ligation (e.g. catalyzed by a ligase enzyme). Ina particular polymerase-based SBS embodiment, fluorescently labelednucleotides are added to a primer (thereby extending the primer) in atemplate dependent fashion such that detection of the order and type ofnucleotides added to the primer can be used to determine the sequence ofthe template. For example, to initiate a first SBS cycle, commands canbe given to deliver one or more labeled nucleotides, DNA polymerase,etc., into/through a flow cell that houses an array of nucleic acidtemplates. The nucleic acid templates may be located at correspondingreaction sites. Those reaction sites where primer extension causes alabeled nucleotide to be incorporated can be detected through an imagingevent. During an imaging event, the illumination system 109 may providean excitation light to the reaction sites. Optionally, the nucleotidescan further include a reversible termination property that terminatesfurther primer extension once a nucleotide has been added to a primer.For example, a nucleotide analog having a reversible terminator moietycan be added to a primer such that subsequent extension cannot occuruntil a deblocking agent is delivered to remove the moiety. Thus, forembodiments that use reversible termination a command can be given todeliver a deblocking reagent to the flow cell (before or after detectionoccurs). One or more commands can be given to effect wash(es) betweenthe various delivery steps. The cycle can then be repeated n times toextend the primer by n nucleotides, thereby detecting a sequence oflength n. Exemplary sequencing techniques are described, for example, inBentley et al., Nature 456:53-59 (2008), WO 2004/018497; U.S. Pat. No.7,057,026; WO 91/06678; WO 2007/123744; U.S. Pat. No. 7,329,492; U.S.Pat. No. 7,211,414; U.S. Pat. No. 7,315,019; U.S. Pat. No. 7,405,281,and US 2008/0108082, each of which is incorporated herein by reference.

For the nucleotide delivery step of an SBS cycle, either a single typeof nucleotide can be delivered at a time, or multiple differentnucleotide types (e.g. A, C, T and G together) can be delivered. For anucleotide delivery configuration where only a single type of nucleotideis present at a time, the different nucleotides need not have distinctlabels since they can be distinguished based on temporal separationinherent in the individualized delivery. Accordingly, a sequencingmethod or apparatus can use single color detection. For example, anexcitation source need only provide excitation at a single wavelength orin a single range of wavelengths. For a nucleotide deliveryconfiguration where delivery results in multiple different nucleotidesbeing present in the flow cell at one time, sites that incorporatedifferent nucleotide types can be distinguished based on differentfluorescent labels that are attached to respective nucleotide types inthe mixture. For example, four different nucleotides can be used, eachhaving one of four different fluorophores. In one embodiment, the fourdifferent fluorophores can be distinguished using excitation in fourdifferent regions of the spectrum. For example, four differentexcitation radiation sources can be used. Alternatively, fewer than fourdifferent excitation sources can be used, but optical filtration of theexcitation radiation from a single source can be used to producedifferent ranges of excitation radiation at the flow cell.

In some embodiments, fewer than four different colors can be detected ina mixture having four different nucleotides. For example, pairs ofnucleotides can be detected at the same wavelength, but distinguishedbased on a difference in intensity for one member of the pair comparedto the other, or based on a change to one member of the pair (e.g. viachemical modification, photochemical modification or physicalmodification) that causes apparent signal to appear or disappearcompared to the signal detected for the other member of the pair.Exemplary apparatus and methods for distinguishing four differentnucleotides using detection of fewer than four colors are described forexample in U.S. Pat. App. Ser. Nos. 61/538,294 and 61/619,878, which areincorporated herein by reference in their entireties. U.S. applicationSer. No. 13/624,200, which was filed on Sep. 21, 2012, is relevant inthis context and also incorporated by reference in its entirety.

The plurality of protocol modules may also include a sample-preparation(or generation) module 137 that is configured to issue commands to thefluidic control system 106 and the temperature control system 110 foramplifying a product within the biosensor 102. For example, thebiosensor 102 may be engaged to the base calling system 100. Theamplification module 137 may issue instructions to the fluidic controlsystem 106 to deliver necessary amplification components to reactionchambers within the biosensor 102. In other embodiments, the reactionsites may already contain some components for amplification, such as thetemplate DNA and/or primers. After delivering the amplificationcomponents to the reaction chambers, the amplification module 137 mayinstruct the temperature control system 110 to cycle through differenttemperature stages according to known amplification protocols. In someembodiments, the amplification and/or nucleotide incorporation isperformed isothermally.

The SBS module 136 may issue commands to perform bridge PCR whereclusters of clonal amplicons are formed on localized areas within achannel of a flow cell. After generating the amplicons through bridgePCR, the amplicons may be “linearized” to make single stranded templateDNA, or sstDNA, and a sequencing primer may be hybridized to a universalsequence that flanks a region of interest. For example, a reversibleterminator-based sequencing by synthesis method can be used as set forthabove or as follows.

Each base calling or sequencing cycle can extend a sstDNA by a singlebase which can be accomplished for example by using a modified DNApolymer ase and a mixture of four types of nucleotides. The differenttypes of nucleotides can have unique fluorescent labels, and eachnucleotide can further have a reversible terminator that allows only asingle-base incorporation to occur in each cycle. After a single base isadded to the sstDNA, excitation light may be incident upon the reactionsites and fluorescent emissions may be detected. After detection, thefluorescent label and the terminator may be chemically cleaved from thesstDNA. Another similar base calling or sequencing cycle may follow. Insuch a sequencing protocol, the SBS module 136 may instruct the fluidiccontrol system I 06 to direct a flow of reagent and enzyme solutionsthrough the biosensor 102. Exemplary reversible terminator-based SBSmethods which can be utilized with the apparatus and methods set forthherein are described in US Patent Application Publication No.2007/0166705 A1, US Patent Application Publication No. 2006/0188901 A1,U.S. Pat. No. 7,057,026, US Patent Application Publication No.2006/0240439 A1, US Patent Application Publication No. 2006/0281109 A1,PCT Publication No. WO 2005/065814, US Patent Application PublicationNo. 2005/0100900 A1, PCT Publication No. WO 2006/064199 and PCTPublication No. WO 2007/010251, each of which is incorporated herein byreference in its entirety. Exemplary reagents for reversibleterminator-based SBS are described in U.S. Pat. No. 7,541,444; U.S. Pat.No. 7,057,026; U.S. Pat. No. 7,414,116; U.S. Pat. No. 7,427,673; U.S.Pat. No. 7,566,537; U.S. Pat. No. 7,592,435 and WO 2007/135368, each ofwhich is incorporated herein by reference in its entirety.

In some embodiments, the amplification and SBS modules may operate in asingle assay protocol where, for example, template nucleic acid isamplified and subsequently sequenced within the same cartridge.

The base calling system 100 may also allow the user to reconfigure anassay protocol. For example, the base calling system 100 may offeroptions to the user through the user interface 114 for modifying thedetermined protocol. For example, if it is determined that the biosensor102 is to be used for amplification, the base calling system 100 mayrequest a temperature for the annealing cycle. Furthermore, the basecalling system 100 may issue warnings to a user if a user has provideduser inputs that are generally not acceptable for the selected assayprotocol.

In embodiments, the biosensor 102 includes millions of sensors (orpixels), each of which generates a plurality of sequences of pixelsignals over successive base calling cycles. Signal processor 130detects the plurality of sequences of pixel signals and attributes themto corresponding sensors (or pixels) in accordance to the row-wiseand/or column-wise location of the sensors on an array of sensors.

Biosensor

FIG. 3 illustrates a cross-section of a biosensor 300 that can be usedin various embodiments. Biosensor 300 has pixel areas 306′, 308′, 310′,312′, and 314′ that can each hold more than one cluster during a basecalling cycle (e.g., 2 clusters per pixel area). Biosensor 300 may havesimilar features as the biosensor 102 (FIG. 1 ) described above and maybe used in, for example, the cartridge. As shown, the biosensor 300 mayinclude a flow cell 302 that is mounted onto a sampling device 304. Inthe illustrated embodiment, the flow cell 302 is affixed directly to thesampling device 304. However, in alternative embodiments, the flow cell302 may be removably coupled to the sampling device 304. The samplingdevice 304 has a sample surface 334 that may be functionalized (e.g.,chemically or physically modified in a suitable manner for conductingthe desired reactions). For example, the sample surface 334 may befunctionalized and may include a plurality of pixel areas 306′, 308′,310′, 312′, and 314′ that can each hold more than one cluster during abase calling cycle (e.g., each having a corresponding cluster pair306AB, 308AB, 310AB, 312AB, and 314AB immobilized thereto). Each pixelarea is associated with a corresponding sensor (or pixel or photodiode)306, 308, 310, 312, and 314, such that light received by the pixel areais captured by the corresponding sensor. A pixel area 306′ can be alsoassociated with a corresponding reaction site 306″ on the sample surface334 that holds a cluster pair, such that light emitted from the reactionsite 306″ is received by the pixel area 306′ and captured by thecorresponding sensor 306. As a result of this sensing structure, in thecase in which two or more clusters are present in a pixel area of aparticular sensor during a base calling cycle (e.g., each having acorresponding cluster pair), the pixel signal in that base calling cyclecarries information based on all of the two or more clusters. As aresult, signal processing as described herein is used to distinguisheach cluster, where there are more clusters than pixel signals in agiven sampling event of a particular base calling cycle.

In the illustrated embodiment, the flow cell 302 includes sidewalls 338,340 and a flow cover 336 that is supported by the sidewalls 338, 340.The sidewalls 338, 340 are coupled to the sample surface 334 and extendbetween the flow cover 336 and the sidewalls 338, 340. In someembodiments, the sidewalls 338, 340 are formed from a curable adhesivelayer that bonds the flow cover 336 to the sampling device 304.

The sidewalls 338, 340 are sized and shaped so that a flow channel 344exists between the flow cover 336 and the sampling device 304. As shown,the flow channel 344 may include a height H₁ that is determined by thesidewalls 338, 340. The height H₁ may be between about 50-400 μm(micrometer) or, more particularly, about 80-200 μm. In the illustratedembodiment, the height H₁ is about 100 μm. The flow cover 336 mayinclude a material that is transparent to excitation light 301propagating from an exterior of the biosensor 300 into the flow channel344. As shown in FIG. 3 , the excitation light 301 approaches the flowcover 336 at a non-orthogonal angle. However, this is only forillustrative purposes as the excitation light 301 may approach the flowcover 336 from different angles.

Also shown, the flow cover 336 may include inlet and outlet ports 342,346 that are configured to fluidically engage other ports (not shown).For example, the other ports may be from the cartridge or theworkstation. The flow channel 344 is sized and shaped to direct a fluidalong the sample surface 334. The height H₁ and other dimensions of theflow channel 344 may be configured to maintain a substantially even flowof a fluid along the sample surface 334. The dimensions of the flowchannel 344 may also be configured to control bubble formation.

As shown in exemplary FIG. 3 , the sidewalls 338, 340 and the flow cover336 are separate components that are coupled to each other. Inalternative embodiments, the sidewalls 338, 340 and the flow cover 336may be integrally formed such that the sidewalls 338, 340 and the flowcover 336 are formed from a continuous piece of material. By way ofexample, the flow cover 336 (or the flow cell 302) may comprise atransparent material, such as glass or plastic. The flow cover 336 mayconstitute a substantially rectangular block having a planar exteriorsurface and a planar inner surface that defines the flow channel 344.The block may be mounted onto the sidewalls 338, 340. Alternatively, theflow cell 302 may be etched to define the flow cover 336 and thesidewalls 338, 340. For example, a recess may be etched into thetransparent material. When the etched material is mounted to thesampling device 304, the recess may become the flow channel 344.

The sampling device 304 may be similar to, for example, an integratedcircuit comprising a plurality of stacked substrate layers 320-326. Thesubstrate layers 320-326 may include abase substrate 320, a solid-stateimager 322 (e.g., CMOS image sensor), a filter or light-management layer324, and a passivation layer 326. It should be noted that the above isonly illustrative and that other embodiments may include fewer oradditional layers. Moreover, each of the substrate layers 320-326 mayinclude a plurality of sub-layers. As will be described in greaterdetail below, the sampling device 304 may be manufactured usingprocesses that are similar to those used in manufacturing integratedcircuits, such as CMOS image sensors and CCDs. For example, thesubstrate layers 320-326 or portions thereof may be grown, deposited,etched, and the like to form the sampling device 304.

The passivation layer 326 is configured to shield the filter layer 324from the fluidic environment of the flow channel 344. In some cases, thepassivation layer 326 is also configured to provide a solid surface(i.e., the sample surface 334) that permits biomolecules or otheranalytes-of-interest to be immobilized thereon. For example, each of thereaction sites may include a cluster of biomolecules that areimmobilized to the sample surface 334. Thus, the passivation layer 326may be formed from a material that permits the reaction sites to beimmobilized thereto. The passivation layer 326 may also comprise amaterial that is at least transparent to a desired fluorescent light. Byway of example, the passivation layer 326 may include silicon nitride(Si3N4) and/or silica (SiO2). However, other suitable material(s) may beused. In the illustrated embodiment, the passivation layer 326 may besubstantially planar. However, in alternative embodiments, thepassivation layer 326 may include recesses, such as pits, wells,grooves, and the like. In the illustrated embodiment, the passivationlayer 326 has a thickness that is about 150-200 nm and, moreparticularly, about 170 nm.

The filter layer 324 may include various features that affect thetransmission of light. In some embodiments, the filter layer 324 canperform multiple functions. For instance, the filter layer 324 may beconfigured to (a) filter unwanted light signals, such as light signalsfrom an excitation light source; (b) direct emission signals from thereaction sites toward corresponding sensors 306, 308, 310, 312, and 314that are configured to detect the emission signals from the reactionsites; or (c) block or prevent detection of unwanted emission signalsfrom adjacent reaction sites. As such, the filter layer 324 may also bereferred to as a light-management layer. In the illustrated embodiment,the filter layer 324 has a thickness that is about 1-5 μm and, moreparticularly, about 3-4 μm. In alternative embodiments, the filter layer324 may include an array of microlenses or other optical components.Each of the microlenses may be configured to direct emission signalsfrom an associated reaction site to a sensor.

In some embodiments, the solid-state imager 322 and the base substrate320 may be provided together as a previously constructed solid-stateimaging device (e.g., CMOS chip). For example, the base substrate 320may be a wafer of silicon and the solid-state imager 322 may be mountedthereon. The solid-state imager 322 includes a layer of semiconductormaterial (e.g., silicon) and the sensors 306, 308, 310, 312, and 314. Inthe illustrated embodiment, the sensors are photodiodes configured todetect light. In other embodiments, the sensors comprise lightdetectors. The solid-state imager 322 may be manufactured as a singlechip through a CMOS-based fabrication processes.

The solid-state imager 322 may include a dense array of sensors 306,308, 310, 312, and 314 that are configured to detect activity indicativeof a desired reaction from within or along the flow channel 344. In someembodiments, each sensor has a pixel area (or detection area) that isabout 1-3 square micrometer (μm²) The array can include 500,000 sensors,5 million sensors, 10 million sensors, or even 130 million sensors. Thesensors 306, 308, 310, 312, and 314 can be configured to detect apredetermined wavelength of light that is indicative of the desiredreactions.

In some embodiments, the sampling device 304 includes a microcircuitarrangement, such as the microcircuit arrangement described in U.S. Pat.No. 7,595,883, which is incorporated herein by reference in theentirety. More specifically, the sampling device 304 may comprise anintegrated circuit having a planar array of the sensors 306,308,310,312,and 314. The array of the sensors 306,308,310,312, and 314 can becommunicatively coupled to a row decoder and a column amplifier ordecoder. The column amplifier can also be communicatively coupled to acolumn analog-to-digital converter (Column ADC/Mux). Other circuitry maybe coupled to the above components, including a digital signal processorand memory. Circuitry formed within the sampling device 304 may beconfigured for at least one of signal amplification, digitization,storage, and processing. The circuitry may collect and analyze thedetected fluorescent light and generate pixel signals (or detectionsignals) for communicating detection data to the signal processor 138.The circuitry may also perform additional analog and/or digital signalprocessing in the sampling device 304. Sampling device 304 may includeconductive vias 330 that perform signal routing (e.g., transmit thepixel signals to the signal processor 138). The pixel signals may alsobe transmitted through electrical contacts 332 of the sampling device304.

However, the sampling device 304 is not limited to the aboveconstructions or uses as described above. In alternative embodiments,the sampling device 304 may take other forms. For example, the samplingdevice 304 may comprise a CCD device, such as a CCD camera, that iscoupled to a flow cell or is moved to interface with a flow cell havingreaction sites therein. In other embodiments, the sampling device 304may be a CMOS-fabricated sensor, including chemically sensitive fieldeffect transistors (chemFET), ion-sensitive field effect transistors(ISFET), and/or metal oxide semiconductor field effect transistors(MOSFET). Such embodiments may include an array of field effecttransistors (FET's) that may be configured to detect a change inelectrical properties within the reaction chambers. For example, theFET's may detect at least one of a presence and concentration change ofvarious analytes. By way of example, the array ofFET's may monitorchanges in hydrogen ion concentration. Such sampling devices aredescribed in greater detail is U.S. Patent Application Publication No.2009/0127589, which is incorporated by reference in the entirety for theuse of understanding such FET arrays.

FIG. 4 shows a cross-section of a biosensor 400 that can be used invarious embodiments. Biosensor 400 has wells 406, 408, 410, 412, and 414that can each hold more than one cluster during a base calling cycle(e.g., 2 clusters per well). The sample surface 334 may be substantiallyplanar as shown in FIG. 3 . However, in alternative embodiments, thesample surface 334 may be shaped to define wells (or reaction chambers)in which each well has one or more reaction sites. The wells may bedefined by, for example, well walls that effectively separate thereaction site(s) of one well from the reaction site(s) of an adjacentwell.

As shown in FIG. 4 , the wells 406, 408, 410, 412, and 414 may bedistributed in a pattern along the sample surface 334. For example, thewells 406, 408, 410, 412, and 414 may be located in rows and columnsalong the sample surface 334 in a manner that is similar to amicroarray. However, it is understood that various patterns of wells406, 408, 410, 412, and 414 may be used. In particular embodiments, eachof the wells 406, 408, 410, 412, and 414 includes more than one clusterof biomolecules (e.g., oligonucleotides) that are immobilized on thesample surface 334. For example, well 406 holds cluster pair 306AB, well408 holds cluster pair 308AB, well 410 holds cluster pair 3 10AB, well412 holds cluster pair 312AB, and well 414 holds cluster pair 314AB.

The sensors are configured to detect light signals that are emitted fromwithin the wells. In particular embodiments, pixel areas 306′, 308′,310′, 312′, and 314′ can be also associated with corresponding wells406, 408, 410, 412, and 414 on the sample surface 334, such that lightemitted from the wells 406, 408, 410, 412, and 414 is received by theassociated pixel areas 306′, 308′, 310′, 312′, and 314′ and captured bythe corresponding sensors 306, 308, 310, 312, and 314.

In embodiments, the sample surface 334 has a fixed position relative tothe sampling device 304 so that the wells 406, 408, 410, 412, and 414have known spatial locations relative to at least one predeterminedsensor (or pixel). The at least one predetermined sensor detectsactivity of the desired reactions from the overlying well. As such, thewells 406, 408, 410, 412, and 414 may be assigned to at least one of thesensors 306, 308, 310, 312, and 314. To this end, the circuitry of thesampling device 304 may include kernels that automatically associatepixel signals (or detection signals) provided by predetermined sensors306, 308, 310, 312, and 314 with the assigned wells 406, 408, 410, 412,and 414. By way of example, when pixel signals are generated by sensor306 shown in FIG. 4 , the pixel signals will automatically be associatedwith the well 406 shown in FIG. 4 . Such a configuration may facilitateprocessing and analyzing the detection data. For instance, the pixelsignals from one well may automatically be located at a certain positionon the array based on row-wise and/or column-wise decoding.

In some embodiments, the sensors (or pixels) are underlying or below theclusters. In other embodiments, the sensors (or pixels) are overlying oron top of the clusters. In yet other embodiments, the sensors (orpixels) are to the side of the clusters (e.g., to the right and/orleft).

Multiple Cluster Base Call Per Sensor (or Pixel)

In embodiments, the technology disclosed increases throughput of thebiosensor 300 by using pixel signals from fewer sensors (or pixels) thana number of clusters base called in a base calling cycle. In particularembodiments, if the biosensor 300 has N active sensors, then thetechnology disclosed uses pixel signals from the N active sensors tobase call N+M clusters, where Mis a positive integer. In embodiments,this is achieved by base calling multiple clusters per sensor (orpixel), as described below.

In embodiments, a sensor (or pixel) on the sample surface 334 isconfigured to receive light emissions from at least two clusters. Insome embodiments, the sensor simultaneously receives the light emissionsfrom the at least two clusters.

In particular embodiments, the intensity of respective light emissionsof the two clusters is significantly different such that one of the twoclusters is a “bright” cluster and the other is a “dim” cluster. Inembodiments, the intensity values vary between base calling cycles andthus the classification of bright and dim can also change betweencycles. In other embodiments, a bright cluster is referred to as a“major” or “dominant” cluster and a dim cluster is referred to as a“minor” or “subordinate” cluster. Some examples of intensity valueratios of emissions between bright and dim clusters include 0.55:0.45,0.60:0.40, 0.65:0.35, 0.70:0.30, 0.75:0.25, 0.80:0.20, 0.85:0.15,0.90:0.10, and 0.95:0.05.

In yet other embodiments, the at least two clusters are not bright anddim clusters, but instead clusters with different intensities orclusters generating different types of signals.

During each sampling event (e.g., each illumination stage or each imageacquisition stage), signal processor 138 receives a common, single pixelsignal for at least two clusters (e.g., both the bright and dimclusters). The common, single pixel generated at each sampling eventincludes/represents/reflects/carries light emissions/intensitysignals/light captured/sensed information for or from the at least twoclusters (e.g., both the bright and dim clusters). In other words, theat least two clusters (e.g., both the bright and dim clusters)contribute to the common, single pixel generated at each sampling event.Accordingly, light emissions from the at least two clusters (e.g., boththe bright and dim clusters) are detected simultaneously at eachsampling event and the common, single pixel reflects light emissionsfrom the at least two clusters (e.g., both the bright and dim clusters).

For example, in FIGS. 3 and 4 , cluster pair 306AB includes two clusters306A and 306B which share a sensor 306. As such, cluster 306A can be thedim cluster and cluster 306B can be the bright cluster, depending ontheir respective intensity values. Signal processor 138 then uses abasecalling algorithm to classify pixel signals from the bright and dimclusters into one of sixteen distributions, as described below. Inparticular embodiments, the bright and dim cluster co-occupy a well,such as well 406. Thus, cluster pairing can be defined based on a sharedpixel area or a shared well, or both.

FIGS. 5A and 5B are scatter plots 500A and 500B that depict base callingof the bright and dim clusters using their respective pixel signalsdetected by the shared sensor in accordance with one embodiment. X-axisof the scatter plots 500A and 500B represents the AT pixel signalsdetected during a second illumination stage of the sampling event whichinduces illumination from a given cluster indicating nucleotide bases Aand T. Y-axis of the scatter plots 500A and 500B represents the CT pixelsignals detected during a first illumination stage of a sampling eventwhich induces illumination from a given cluster indicating nucleotidebases C and T.

Scatter plot 500A shows four distributions 502, 504, 506, and 508 towhich signal processor 138 classifies pixel signals from the brightcluster. In the illustrated embodiment, distribution 502 representsnucleotide base C in the bright cluster, distribution 504 representsnucleotide base T in the bright cluster, distribution 506 representsnucleotide base Gin the bright cluster, and distribution 508 representsnucleotide base A in the bright cluster.

Scatter plot 500B shows sixteen sub-distributions (or distributions)502A-D, 504A-D, 506A-D, and 508A-D, with four sub-distributions for eachof the four distributions 502, 504, 506, and 508 of the scatter plot500A), to which signal processor 138 classifies pixel signals from thedim cluster. In the illustrated embodiment, sub-distributions annotatedwith letter “A” represent nucleotide base C in the dim cluster,sub-distributions annotated with letter “B” represent nucleotide baseTin the dim cluster, sub-distributions annotated with letter “C”represent nucleotide base Gin the dim cluster, and sub-distributionsannotated with letter “D” represent nucleotide base A in the dimcluster. In other embodiments, different encodings of the bases may beused. When the signal processor classifies pixel signals from a dimcluster in one of the sixteen sub-distributions, the classification ofthe corresponding bright cluster is determined by the distribution whichincludes the dim cluster's sub-distribution. For example, if a dimcluster is classified to sub-distribution 508B (nucleotide base T), thenthe distribution for the corresponding bright cluster is 508 (nucleotidebase A). As a result, the signal processor 138 base calls the brightcluster as A and the dim cluster as T.

FIG. 6 is a scatter plot 600 that depicts sixteen distributions (orbins) produced by intensity values from bright and dim clusters of acluster pair in accordance with one embodiment. In embodiments, thesixteen bins are produced over a plurality of base calling cycles.Signal processor 138 combines pixel signals from the bright and dimclusters and maps them into one of the sixteen bins. When the combinedpixel signals are mapped to bin 612 for a base calling cycle, the signalprocessor 138 base calls the bright cluster as C and the dim cluster asC. When the combined pixel signals are mapped to bin 614 for the basecalling cycle, the signal processor 138 base calls the bright cluster asC and the dim cluster as T. When the combined pixel signals are mappedto bin 616 for the base calling cycle, the signal processor 138 basecalls the bright cluster as C and the dim cluster as G. When thecombined pixel signals are mapped to bin 618 for the base calling cycle,the signal processor 138 base calls the bright cluster as C and the dimcluster as A

When the combined pixel signals are mapped to bin 622 for the basecalling cycle, the signal processor 138 base calls the bright cluster asT and the dim cluster as C. When the combined pixel signals are mappedto bin 624 for the base calling cycle, the signal processor 138 basecalls the bright cluster as T and the dim cluster as T. When thecombined pixel signals are mapped to bin 626 for the base calling cycle,the signal processor 138 base calls the bright cluster as T and the dimcluster as G. When the combined pixel signals are mapped to bin 628 forthe base calling cycle, the signal processor 138 base calls the brightcluster as T and the dim cluster as A

When the combined pixel signals are mapped to bin 632 for the basecalling cycle, the signal processor 138 base calls the bright cluster asG and the dim cluster as C. When the combined pixel signals are mappedto bin 634 for the base calling cycle, the signal processor 138 basecalls the bright cluster as G and the dim cluster as T. When thecombined pixel signals are mapped to bin 636 for the base calling cycle,the signal processor 138 base calls the bright cluster as G and the dimcluster as G. When the combined pixel signals are mapped to bin 638 forthe base calling cycle, the signal processor 138 base calls the brightcluster as G and the dim cluster as A

When the combined pixel signals are mapped to bin 642 for the basecalling cycle, the signal processor 138 base calls the bright cluster asA and the dim cluster as C. When the combined pixel signals are mappedto bin 644 for the base calling cycle, the signal processor 138 basecalls the bright cluster as A and the dim cluster as T. When thecombined pixel signals are mapped to bin 646 for the base calling cycle,the signal processor 138 base calls the bright cluster as A and the dimcluster as G. When the combined pixel signals are mapped to bin 648 forthe base calling cycle, the signal processor 138 base calls the brightcluster as A and the dim cluster as A

FIG. 7A is a detection table 700A that illustrates a base calling schemefor one dye and two illumination stage sequencing protocol in accordancewith one embodiment. One avenue of differentiating between the differentstrategies for detecting nucleotide incorporation in a sequencingreaction using one fluorescent dye (or two or more dyes of same orsimilar excitation/emission spectra) is by characterizing theincorporations in terms of the presence or relative absence, or levelsin between, of fluorescence transition that occurs during a sequencingcycle. As such, sequencing strategies can be exemplified by theirfluorescent profile for a sequencing cycle. For strategies disclosedherein, “1” and “0” denotes a fluorescent state in which a nucleotide isin a signal state (e.g., detectable by fluorescence) or whether anucleotide is in a dark state (e.g., not detected or minimally detectedat an imaging step). A “0” state does not necessarily refer to a totallack, or absence of signal. Minimal or diminished fluorescence signal(e.g., background signal) is also contemplated to be included in thescope of a “0” state as long as a change in fluorescence from the firstto the second illumination event (or vice versa) can be reliablydistinguished. In one embodiment, an exemplary strategy for detectingand determining nucleotide incorporation in a sequencing reaction usingone fluorescent dye (or two dyes of same or similar excitation/emissionspectra) and two illumination events is exemplified by the detectiontable 700A.

In the illustrated embodiment, during the first illumination stage (ATsignal), nucleotide base A is labeled or on (depicted by bit 1),nucleotide base C is unlabeled or off (depicted by bit 0), nucleotidebase G is unlabeled or off (depicted by bit 0), and nucleotide base Tislabeled or on (depicted by bit 1). During the second illumination stage(CT signal), nucleotide base A is unlabeled or off (depicted by bit 0),nucleotide base C is labeled or on (depicted by bit 1), nucleotide baseGis unlabeled or off (depicted by bit 0), and nucleotide base T islabeled or on (depicted by bit 1).

FIG. 7B is a base calling table 700B that shows a classification schemefor classifying combined pixel signals, each pixel signal includinginformation from the bright and dim clusters of a cluster pair, into oneof sixteen bins in accordance with one embodiment.

The technology disclosed generates a pixel signal that representsinformation sensed from all of the multiple clusters in a pixel area ofa shared sensor. A sequence of such pan-cluster pixel signals is thenmapped to bins to base call all the clusters. Thus, a separate, discretepixel signal for each cluster is not generated. This has the advantageof multifold reduction in image acquisition and thereby reducingsequencing time and accelerating sequence processing.

Consider FIG. 7B in which a bright cluster and a dim cluster in a pixelarea are base called. At each cycle, two pixels signals are sampled: anAT signal and a CT signal. During the first sampling event, lightemissions from both the bright and dim clusters for fluorescentlylabeled adenines (A) and thymines (T) are recorded in the AT signal, asopposed to two separate AT signals, i.e., one for the bright cluster andanother for the dim cluster. Similarly, during the second samplingevent, light emissions from both the bright and dim clusters forfluorescently labeled cytosines (C) and thymines (T) are recorded in theCT signal, as opposed to two separate CT signals, i.e., one for thebright cluster and another for the dim cluster.

This way, light emissions from both clusters are received during asingle sampling event and yield a common, single pixel signal.Therefore, for each sampling event, emissions from both the bright anddim clusters are jointly represented in a common, single pixel signal.

Furthermore, a common, single sequence of pixel signals is used tojointly base call both the bright and dim clusters at each cycle. InFIG. 7B, the AT and CT signals together form the common, single sequenceof pixel signals. Thus, the technology disclosed does not use twoseparate sequences of pixel signals, i.e., one for the bright clusterand another for the dim cluster, to separately base call the bright anddim clusters. This has the advantage of multifold reduction in signalprocessing and thereby reducing sequencing time and acceleratingsequence processing.

The disclosed base calling involves mapping the common, single sequenceof pixel signals to bins. For instance, in FIG. 7B, with values 1 and 0,the sequence of AT and CT signals is mapped to bin 1 and the bright anddim clusters are assigned base calls A and A, respectively.

In the example shown in FIG. 7B, a deterministic bright to dim clusterintensity ratio of 0.7:0.3 is used. In embodiments, the intensity ratiois undetermined, as such, it produces detectable bright and dim clustersthat share a pixel area or share a well, or both.

As a result of the intensity ratio being 0.7:0.3 (i.e., intensity valuesof light emissions from the bright and dim clusters being significantlydifferent), the pixel signals readout from the shared sensor during thetwo illumination stages over a plurality of base calling cycles producessixteen bins 701 (bins 1-16). Each bin has a unique pair of pixel signalvalues (e.g., unique pair 710 for bin 1), the pair comprises a firstpixel signal value 706 for the two clusters in the first illuminationstage (AT signal) and a second pixel signal value 708 for the twoclusters in the second illumination stage (CT signal).

Each pixel signal value 706 or 708 is in tum composed of two signalportions 706A and 706B or 708A and 708B, which are additively combinedto produce the corresponding pixel signal values 706 or 708. Thus, acommon, single pixel signal is generated both the bright and dimclusters.

For each pixel signal value 706 or 708, a first signal portion 706A or708A is determined from the intensity value of light emissions by thefirst cluster and a second signal portion 706B or 708B is determinedfrom the intensity value of light emissions by the second cluster. Inthe example shown in base calling table 700B, the first cluster is thebright cluster 702 and the second cluster is the dim cluster 704.

Since the intensity ratio is 0.7:0.3, the first and second pixel signalscan take one of the four possible values—1, 0, 0.7, or 0.3.Additionally, when the bright cluster produces an “on” bit, itscontribution or signal portion (706A, 708A) is 0.7. In contrast, whenthe dim cluster produces an “on” bit, its contribution or signal portion(706B, 708B) is 0.3. A contribution or signal portion representing an“off” bit is identified by 0 for both the clusters. Sixteen uniquecombinations of the four possible values 1, 0, 0.7, and 0.3 produce thesixteen bins 701.

Once the sixteen bins 701 are identified by the signal processor 138 fora bright-dim cluster pair overlying a shared sensor or well over aplurality of base calling cycles, the signal processor 138 uses the basecalling table 700B to base call the bright and dim cluster in successivebase calling cycles. In one embodiment, the identification results inclassification of the well as holding more than one cluster (i.e., thebright cluster and the dim cluster). Thus, in a successive base callingcycle, the signal processor performs a first pixel readout of the sharedsensor for the first illumination stage (AT signal). This first pixelreadout produces a first pixel signal. Similarly, a second pixel readoutfor the second illumination stage (CT signal) produces a second pixelsignal. The first and second pixel signals produce intensity values thatare combined to form a value pair. This value pair can be comparedagainst one of the sixteen unique value pairs in the base calling table700B. Based on the comparison, one of the sixteen bins is selected. Basecall for the bright and dim clusters is made in accordance with thenucleotide bases assigned to the selected bin. This process is repeatedfor subsequent base calling cycles to identify nucleotide bases presentin the respective nucleotide sequences of the bright and dim cluster.

Therefore, the technology disclosed treats emissions from all theclusters as useful for base calling, irrespective of their relativestrength. This is because the clusters that have weaker emissions (e.g.,dim cluster) are not separately base called; instead they are jointlybase called with clusters that have stronger emissions (e.g., brightcluster) using a common, single sequence of pixel signals carrying boththe stronger and weaker em1ss10ns.

As discussed above, the shared sensor captures photons from twodifferent clusters (e.g., a bright cluster and a dim cluster). In someembodiments, the signal portions are detected by deconvoluting thesignal readings from the shared sensor to distinguish the individualsignal portions generated by each of the clusters.

FIG. 8 shows a method 800 of base calling by analyzing pixel signalsemitted by a plurality of clusters that share a pixel area in accordancewith one embodiment. At action 802, a first pixel signal that representslight gathered from multiple clusters in a first pixel area during afirst illumination stage of the base calling cycle is detected. In someembodiments, the first pixel area receives light from an associated wellon the sample surface 334. In other embodiments, the first pixel areareceives light from more than one associated well on the sample surface334.

At action 804, a second pixel signal that represents light gathered frommultiple clusters in the first pixel area during a second illuminationstage of the base calling cycle is detected.

In embodiments, the first pixel area underlies a plurality of clustersthat shares the first pixel area. The first and second pixel signals canbe gathered by a first sensor from the first pixel area. The first andsecond pixel signals can be detected by the signal processor 138, whichis configured for processing pixel signals gathered by the first sensor.

In some embodiments, the first illumination stage can induceillumination from the first and second clusters to produce emissionsfrom labeled nucleotide bases A and T, and the second illumination stagecan induce illumination from the first and second clusters to produceemissions from labeled nucleotide bases C and T.

At action 806, a combination of the first and second pixel signals isused to identify nucleotide bases incorporated onto each cluster of theplurality of clusters during the base calling cycle. In embodiments,this includes mapping the first pixel signal into at least four bins andmapping the second pixel signal into at least four bins, and combiningthe mapping of the first and second pixel signals for the base calling.

In embodiments, method 800 is applied to identify the nucleotide basesincorporated onto the plurality of clusters at a plurality of pixelareas during the base calling cycle. In embodiments, method 800 isrepeated over successive base calling cycles to identify the nucleotidebases incorporated onto the plurality of clusters at the plurality ofpixel areas during each of the base calling cycles.

In some embodiments, for each of the base calling cycles, the first andsecond pixel signals emitted by the plurality of clusters at theplurality of pixel areas are detected and stored. After the base callingcycles, the combination of the first and second pixel signals is used toidentify the nucleotide bases incorporated onto the plurality ofclusters at the plurality of pixel areas during each of the previousbase calling cycles.

FIG. 9 depicts a method 900 of identifying pixel areas with more thanone cluster on the sample surface 334 of the biosensor 300 and basecalling clusters at the identified pixel areas in accordance with oneembodiment. At action 902, a plurality of base calling cycles isperformed. Each base calling cycle has a first illumination stage and asecond illumination stage.

At action 904, a sensor associated with a pixel area of the samplesurface 334 captures—(a) a first set of intensity values generatedduring the first illumination stage of the base calling cycles and (b) asecond set of intensity values generated during the second illuminationstage of the base calling cycles. In embodiments, the intensity valuesare normalized. Also, in some embodiments, the pixel area receives lightfrom an associated well on the sample surface 334.

At action 906, the signal processor 138 fits (shown in FIG. 6 ) to thefirst and second sets of intensity values to a one of a set ofdistributions (where a distribution is a area in the two dimensionalplot of FIG. 6 ), including sixteen distributions in this example. and,based on the fitting, classifies the pixel area as having more than onecluster. In embodiments, the signal processor 138 uses one or morealgorithms for fitting the sixteen distributions. Examples of algorithmsinclude k-means clustering algorithm, k-means-like clustering algorithm,expectation maximization algorithm, and histogram based algorithm.

At action 908, for a successive base calling cycle, the signal processor138 detects the first and second sets of intensity values for a clustergroup at the pixel area. At action 910, the signal processor 138 selectsa distribution for the cluster group from among the sixteendistributions. The distribution identifies a nucleotide base present ineach cluster of the cluster group.

In some embodiments, the intensity ratios are an inherent property ofthe bright and dim clusters that produce significantly different lightemissions. In other embodiments, the intensity ratios and thesignificantly different light emissions between clusters are actuated bythe following embodiments, such as uneven distribution of clusters on aflat surface, dual wells per sensor (or pixel), and off-axisillumination.

Flat Surface-Based Spatial Analysis of Unevenly Distributed Clusters

FIG. 10 illustrates atop plan view 1000 of the sample surface 334 havingpixel areas (depicted as rectangles) on which a plurality of clusters(depicted as circles) are unevenly distributed in accordance with oneembodiment. Positions of the clusters on the surface well 334 may not beconfined by wells relative to the locations of the sensors (or pixels).Such arrangement of clusters on the sample surface 334 is referred to asuneven distribution. In particular embodiments, the clusters areunevenly distributed on a “flat” configuration of the sample surface 334that does not include wells. In such a flat surface embodiment, thepixel areas can overlap.

In the illustrated embodiment, consider two example clusters 1002 and1004 that share four pixel areas A, B, C, and D. Depending on thecluster's relative position with respect to centers of the pixel areasA, B, C, and D, the corresponding sensors (or pixels) receive differentamount of light emissions. This produces illumination patterns thatcreate differential crosstalk between the clusters I 002 and I 004 overa plurality of base calling cycles of a sequencing run, which can beused to construct a map of cluster locations on the sample surface 334,as described below. The differential crosstalk is embodied in the pixelsignals as information from two or more clusters in one pixel signal.

Signal processor 138 executes time sequence and spatial analysis of aplurality of sequences of pixel signals for the clusters to detectpatterns of illumination corresponding to individual clusters unevenlydistributed on the sample surface 334. The plurality of sequences ofpixel signals encodes differential crosstalk between at least twoclusters resulting from their uneven distribution over the pixel areas.

Spatial analysis includes using the sequences of pixel signals gatheredfrom a group of pixel areas to determine spatial characteristics of agiven cluster, including location of the given cluster on the samplesurface 334. After the cluster locations and their illumination patternsare identified over the plurality of base calling cycles, the clusterscan be base called by the signal processor 138 using one of thesequencing protocols discussed above.

In the spatial analysis embodiment, the technology disclosed increasesthroughput of the biosensor 300 by using N sensors (or pixels) to locateand base call N+M unevenly distributed clusters on the sample surface334, where Mis a positive integer. In some embodiments, M is equal to Nor almost equal to N. In other embodiments, when two clusters, whichshare (or co-occupy) a pixel area and/or well, are not separatelydetectable due to inadequate difference in intensity values, M might notbe equal to N or even be less than N.

Dual Wells Per Sensor (or Pixel)

FIG. 1 lA illustrates a side view 1100A of a sample surface having twowells per pixel area including a dominant (or major) well and asubordinate (or minor) well in accordance with one embodiment. FIG. 11Bdepicts a top plan view 1100B of the sample surface of FIG. 11A.

In the illustrated embodiment, shared sensor 1106 (or pixel) correspondsto two wells 1102 and 1104 on the sample surface 334. The dominant wellhas a larger cross section over the pixel area than the subordinatewell. Well 1104 is the dominant well and well 1102 is the subordinatewell because well 1104 has a larger cross section over the sensor 1106.

In embodiments, the two wells have different offsets relative to acenter of the pixel area 1106′. In the illustrated embodiment, dominantwell 1104 is more proximate to the pixel area center 1106A than thesubordinate well 1102 (i.e., dominant well 1104 has a smaller offsetrelative to the pixel area center 1106A than the subordinate well 1102).

Due to the differential cross section coverage and relative offsetsresult, the sensor 1106 receives different amounts of illumination fromthe two wells during illumination stages of the base calling cycle (orsampling event). Since each of the wells 1102 and 1104 holds acorresponding cluster 1102A and 1104A, the different amounts ofillumination allow for identification of one of the clusters as bright(or major) and the other as dim (or minor). In the illustratedembodiment, cluster 1102A within the dominant well 1102 is identified asthe bright cluster and cluster 1104A within the subordinate well 1104 isidentified as the dim cluster. In embodiments, sensor 1106 receives anamount of illumination from the bright cluster 1102A that is greaterthan an amount of illumination received from the dim cluster 1104A inthe subordinate well 1104.

After the bright and dim clusters are identified, they can be basecalled by the signal processor 138 using one of the sequencing protocolsdiscussed above. In some dual well per sensor (or pixel) embodiments,the technology disclosed increases throughput of the biosensor 300 bybase calling two clusters 1102A and 1102B held by two correspondingwells 1102 and 1104 using one shared sensor 1106. In other dual well persensor (or pixel) embodiments, the technology disclosed increasesthroughput of the biosensor 300 by using N sensors to base call N+Mclusters on corresponding N+M wells of the sample surface 334, where Misa positive integer. In some embodiments, M is equal to N or almost equalto N. In other embodiments, M might not be equal to N or even be lessthan N.

Off-Axis Illumination

FIGS. 12A and 12B show off-axis illumination 1200A and 1200B of a welloverlying a pixel area of a sample surface. Illumination system 109 isconfigured to illuminate the pixel areas 1204′ and 1214′ (associatedwith sensors 1204 and 1214) with different angles of illuminationsignals 1201 and 1211 during illumination stages of a base callingcycle. As a result, wells 1202 and 1212 are illuminated with off-axis ornon-orthogonal illumination signals. This produces asymmetricallyilluminated well regions in each of the wells 1202 and 1212, depicted inFIGS. 12A and 12B with light and dark shaded areas in each well. Theasymmetrically illuminated well regions of a well include at least adominant well region 1202B′ or 1212A′ (depicted in lighter shade) and asubordinate well region 1202A′ or 1212 B′ (depicted in darker shade),such that during the base calling cycle the dominant well region isilluminated more than the subordinate well region.

Each well is configured to hold more than one cluster during the basecalling cycle, with the dominant and subordinate well regions eachincluding a cluster. In the illustrated embodiment, well 1202 holds twoclusters 1202A and 1202B, with cluster 1202A within the subordinate wellregion 1202A′ and cluster 1202B within the dominant well region 1202B′.Well 1212 holds two clusters 1212A and 1212B, with cluster 1212A withinthe dominant well region 1212A′ and cluster 1212B within the subordinatewell region 1202B′.

Due to the off-axis illumination, pixel areas 1204′ and 1214′ of thewells 1202 and 1212 receive different amounts of illumination fromdominant and subordinate regions of a well. As a result, during the basecalling cycle, clusters in the dominant well regions produce greateramounts of illumination than clusters in the subordinate well regions.For each well, this allows for identification of one of the clusters asbright (or major) and the other as dim (or minor). In the illustratedembodiment, for well 1202, cluster 1202B within the dominant well region1202B′ is identified as the bright cluster and cluster 1202A within thesubordinate well region 1202A′ is identified as the dim cluster. Forwell 1212, cluster 1212A within the dominant well region 1212A′ isidentified as the bright cluster and cluster 1212B within thesubordinate well region 1212B′ is identified as the dim cluster.

After the bright and dim clusters are identified for each well, they canbe base called by the signal processor 138 using one of the sequencingprotocols discussed above. In the off-axis illumination embodiment, thetechnology disclosed increases throughput of the biosensor 300 by usingN sensors (or pixels) to base call N+M clusters within Nnon-orthogonally illuminated wells on the sample surface 334, where Misa positive integer. In some embodiments, Mis equal to Nor almost equalto N. In other embodiments, when two clusters, which share (orco-occupy) a pixel area and/or well, are not separately detectable dueto inadequate difference in intensity values, M might not be equal to Nor even be less than N.

In one embodiment, the off-axis illumination is at a forty-five degreeangle. In some embodiments, one well overlies per pixel area. In otherembodiments, two wells overlie per pixel area.

FIG. 12C illustrates asymmetrically illuminated well regions 1200Cproduced by the off-axis illumination of FIGS. 12A and 12B in accordancewith one embodiment. As shown in FIG. 12C, well region 1220 is moreilluminated than well region 1230.

Clauses

The disclosure also includes the following clauses:

-   1. A device for base calling, comprising:

a receptacle and a biosensor, the receptacle holding the biosensor, thebiosensor having

-   -   a sample surface that holds a plurality of clusters during a        sequence of sampling events,    -   an array of sensors configured to generate a plurality of        sequences of pixel signals, the array having a number N of        active sensors, the sensors in the array disposed relative to        the sample surface to generate respective pixel signals during        the sequence of sampling events from the number N of        corresponding pixel areas of the sample surface to produce the        plurality of sequences of pixel signals, and    -   a communication port which outputs the plurality of sequences of        pixel signals; and

a signal processor coupled to the receptacle, and configured to receiveand to process the plurality of sequences of pixel signals to classifyresults of the sequence of sampling events on clusters in the pluralityof clusters, including using the plurality of sequences of pixel signalsto classify results of the sequence of sampling events on a number N+Mof clusters in the plurality of clusters from the number N of activesensors, where Mis a positive integer.

-   2. The device of clause 1, wherein the results of the sequence of    sampling events correspond to nucleotide bases in the clusters.-   3. The device of clause I or clause 2, wherein the sampling events    comprise two illumination stages in time sequence, and sequences of    pixel signals in the plurality of sequences of pixel signals include    a set of signal samples for each sampling event, the set including    at least one pixel signal from each of the two illumination stages.-   4. The device of clause 3, wherein the signal processor includes    logic to classify results for two clusters from the sequences of    pixel signals from a single sensor in the array of sensors.-   5. The device of clause 4, wherein the logic to classify results for    two clusters includes mapping a first pixel signal of the set of    signal samples for a sampling event from a particular sensor into at    least four bins, and mapping a second pixel signal of the set of    signal samples for the sampling event into at least four bins, and    logically combining the mapping of the first and second pixel    signals to classify the results for two clusters.-   6. The device of any one of clauses I to 5, wherein the sensors in    the array of sensors comprise light detectors.-   7. The device of any one of clauses I to 6, wherein the sampling    events comprise two illumination stages in time sequence, and    sequences of pixel signals in the plurality of sequences of pixel    signals include a set of signal samples for each sampling event, the    set including at least one pixel signal from each of the two    illumination stages, and wherein the first illumination stage    induces illumination from a given cluster indicating nucleotide    bases A and T and the second illumination stage induces illumination    from a given cluster indicating nucleotide bases C and T, and said    classifying results comprises calling one of the nucleotide bases A,    C, Tor G.-   8. The device of any one of clauses 1 to 7, wherein the sample    surface holds clusters that are distributed unevenly over the pixel    areas, and the signal processor executes time sequence and spatial    analysis of the plurality of sequences of pixel signals to detect    patterns of illumination corresponding to individual clusters on the    sample surface, and to classify the results of the sampling events    for the individual clusters, wherein the plurality of sequences of    pixel signals encodes differential crosstalk between at least two    clusters resulting from their uneven distribution over the pixel    areas.-   9. The device of any one of clauses 1 to 8, wherein the sample    surface comprises an array of wells overlying the pixel areas,    including two wells per pixel area, the two wells per pixel area    including a dominant well and a subordinate well, the dominant well    having a larger cross section over the pixel area than the    subordinate well.-   10. The device of any one of clauses 1 to 9, wherein the sample    surface comprises an array of wells overlying the pixel areas, and    the sampling events include at least one chemical stage with a    number K of illumination stages where K is a positive integer, where    the illumination stages of the K illumination stages illuminate the    pixel areas with different angles of illumination, and the sequences    of pixel signals include a set of signal samples for each sampling    event, the set including the number K of pixel signals for the at    least one chemical stage of the sampling events.-   11. The device of any one of clauses 1 to 10, wherein the sample    surface comprises an array of wells overlying the pixel areas, and    the sampling events include a first chemical stage with a number K    of illumination stages where K is a positive integer, where the    illumination stages of the K illumination stages illuminate the    pixel areas with different angles of illumination, and a second    chemical stage with a number J of illumination stages where J is a    positive integer, where the illumination stages of the K    illumination stages in the first chemical stage and of the J    illumination stages in the second chemical stage illuminate the    wells in the array of wells with different angles of illumination,    and the sequences of pixel signals include a set of signal samples    for each sampling event, the set including the number K of pixel    signals for the first chemical stage plus the number J of pixel    signals for the second chemical stage of the sampling events.-   12. A biosensor for base calling, comprising:

a sampling device, the sampling device including a sample surface havingan array of pixel areas and a solid-state imager having an array ofsensors, each sensor generating pixel signals in each base callingcycle, each pixel signal representing light gathered from acorresponding pixel area of the sample surface; and

a signal processor configured for connection to the sampling device thatreceives and processes the pixel signals from the sensors for basecalling in a base calling cycle, and uses the pixel signals from fewersensors than a number of clusters base called in the base calling cycle.

-   13. The biosensor of clause 12, wherein a pixel area receives light    from a well on the sample surface and the well is configured to hold    more than one cluster during the base calling cycle.-   14. The biosensor of clause 13, wherein a cluster comprises a    plurality of single-stranded deoxyribonucleic acid (abbreviated DNA)    fragments having an identical nucleic acid sequence.-   15. A computer-implemented method of base calling, including:

for a base calling cycle of a sequencing by synthesis (abbreviated SBS)run, receiving from a communication port

-   -   a plurality of sequences of pixel signals, the plurality of        sequences of pixel signals being generated by an array of        sensors, the array having a number N of active sensors, the        sensors in the array disposed relative to the sample surface to        generate respective pixel signals during the sequence of        sampling events from the number N of corresponding pixel areas        of the sample surface to produce the plurality of sequences of        pixel signals; and

processing the plurality of sequences of pixel signals to classifyresults of the sequence of sampling events on clusters in the pluralityof clusters, including using the plurality of sequences of pixel signalsto classify results of the sequence of sampling events on clusters inthe plurality of clusters, including using the plurality of sequences ofpixel signals to classify results of the sequence of sampling events ona number N+M of clusters in the plurality of clusters from the number Nof active sensors, where Mis a positive integer.

-   16. The method of clause 15, further including:

mapping a first pixel signal, which represents light gathered from afirst pixel area during a first illumination stage of the base callingcycle, into at least four bins and mapping a second pixel signal, whichrepresents light gathered from the first pixel area during a secondillumination stage of the base calling cycle, into at least four bins,and

combining the mapping of the first and second pixel signals to identifythe incorporated nucleotide bases.

-   17. The method of clause 15 or clause 16, further including applying    the method to identify the nucleotide bases incorporated onto the    plurality of clusters at a plurality of pixel areas during the base    calling cycle.-   18. The method of clause 17, further including repeating the method    over successive base calling cycles to identify the nucleotide bases    incorporated onto the plurality of clusters at the plurality of    pixel areas during each of the base calling cycles.-   19. The method of clause 18, further including:

for each of the base calling cycles, detecting and storing the first andsecond pixel signals emitted by the plurality of clusters at theplurality of pixel areas, and

after the base calling cycles, using the combination of the first andsecond pixel signals to identify the nucleotide bases incorporated ontothe plurality of clusters at the plurality of pixel areas during each ofthe previous base calling cycles.

-   20. The method of any one of clauses 16 to 19, wherein the first    pixel area receives light from an associated well on a sample    surface.-   21. The method of clause 20, wherein the first pixel area receives    light from more than one associated well on the sample surface.-   22. The method of any one of clauses 16 to 21, wherein the first and    second pixel signals are gathered by a first sensor from the first    pixel area.-   23. The method of clause 22, wherein the first and second pixel    signals are detected by a signal processor configured for processing    pixel signals gathered by the first sensor.-   24. The method of any one of clauses 15 to 23, wherein the first    illumination stage induces illumination from the first and second    clusters to produce emissions from labeled nucleotide bases A and T    and the second illumination stage induces illumination from the    first and second clusters to produce emissions from labeled    nucleotide bases C and T.-   25. The method of any one of clauses 15 to 24, in which said base    calling includes using a device as defined in any one of clauses 1    to 11.-   26. A method of identifying pixel areas with more than one cluster    on a sample surface of a biosensor and base calling clusters at the    identified pixel areas, including:

performing a plurality of base calling cycles, each base calling cyclehaving a first illumination stage and a second illumination stage;

capturing at a sensor associated with a pixel area of the samplesurface,

-   -   a first set of intensity values generated during the first        illumination stage of the base calling cycles, and    -   a second set of intensity values generated during the second        illumination stage of the base calling cycles;

fitting sixteen distributions to the first and second sets of intensityvalues using a signal processor and, based on the fitting, classifyingthe pixel area as having more than one cluster; and

for a successive base calling cycle,

-   -   detecting the first and second sets of intensity values for a        cluster group at the pixel area using the signal processor, and    -   selecting a distribution for the cluster group, wherein the        distribution identifies a nucleotide base present in each        cluster of the cluster group.

-   27. The method of clause 26, wherein the fitting comprises using one    or more algorithms, including a k-means clustering algorithm, a    k-means-like clustering algorithm, an expectation maximization    algorithm, and a histogram based algorithm.

-   28. The method of clause 26 or clause 27, further including    normalizing the intensity values.

-   29. The method of any one of clauses 26 to 28, wherein the pixel    area receives light from an associated well on the sample surface.

-   30. The method of any one of clauses 26 to 29, in which said    identifying and base calling includes using a device as defined in    any one of clauses 1 to 11 or a biosensor as defined in any one of    clauses 12 to 14.

-   31. A computer-implemented method of base calling, comprising:

providing a first pixel signal that represents light gathered from afirst pixel area during a first illumination stage of a base callingcycle of a sequencing by synthesis (abbreviated SBS) run and a secondpixel signal that represents light gathered from said first pixel areaduring a second illumination stage of said base calling cycle of saidSBS run, wherein the first pixel area underlies first and secondclusters that share the first pixel area;

providing a signal processor configured for processing at least saidfirst and second pixel signals;

mapping the first pixel signal into at least four bins and mapping thesecond pixel signal into at least four bins using said signal processor;and

logically combining the mapping of the first and second pixel signals toidentify the nucleotide base incorporated onto each of said first andsecond clusters during said base calling cycle.

-   32. A computer-implemented method of identifying pixel areas with    more than one cluster on a sample surface of a biosensor and base    calling clusters at the identified pixel areas, compnsmg:

providing a first set of intensity values generated during a firstillumination stage of a base calling cycle and a second set of intensityvalues generated during a second illumination stage of the base callingcycle, wherein the first and second sets of intensity values representthe intensity of light gathered at a sensor associated with a pixel areaof the sample surface;

fitting sixteen distributions to the first and second sets of intensityvalues using a signal processor and, based on the fitting, classifyingthe pixel area as having more than one cluster; and

for a successive base calling cycle,

-   -   providing first and second sets of intensity values for a        cluster group at the pixel area using the signal processor, and    -   selecting a distribution for the cluster group, wherein the        distribution identifies a nucleotide base present in each        cluster of the cluster group.

-   33. The computer-implemented method of clause 32, wherein the    fitting comprises using one or more algorithms, including a k-means    clustering algorithm, a k-means-like clustering algorithm, an    expectation maximization algorithm, and a histogram based algorithm.

-   34. A device for base calling, comprising:

a receptacle and a biosensor, the receptacle holding the biosensor, thebiosensor having

-   -   a sample surface configured to hold a plurality of clusters        during a sequence of sampling events, the sample surface        comprising a number N of pixel areas, and the sampling events        comprising two illumination stages in time sequence,    -   an array of sensors comprising light detectors configured to        generate a plurality of sequences of pixel signals including at        least one pixel signal for each pixel area and illumination        stage, the array having a number N of active sensors each        associated with a corresponding pixel area of the N pixel areas        and configured to detect light emissions gathered from the        associated pixel area, to generate respective pixel signals        during the sequence of sampling events representing the light        emissions gathered from the corresponding pixel area to produce        the plurality of sequences of pixel signals, wherein the sample        surface is configured such that at least one active sensor        detects light emissions from at least two clusters forming a        cluster pair of the plurality of clusters, wherein the intensity        of respective light emissions of the two clusters is        significantly different, and    -   a communication port which outputs the plurality of sequences of        pixel signals; and

a signal processor coupled to the receptacle, and configured to receiveand to process the plurality of sequences of pixel signals to classifyresults of the sequence of sampling events on clusters in the pluralityof clusters, including using the plurality of sequences of pixel signalsto classify results of the sequence of sampling events on a number N+Mof clusters in the plurality of clusters from the number N of activesensors, where Mis a positive integer, by classifying results for thetwo clusters forming a cluster pair from the sequences of pixel signalsfrom the at least one active sensor in the array of sensors.

-   35. The device of clause 34, wherein the results of the sequence of    sampling events correspond to nucleotide bases in the clusters,    preferably wherein the first illumination stage induces illumination    from a given cluster indicating nucleotide bases A and T and the    second illumination stage induces illumination from a given cluster    indicating nucleotide bases C and T, and said classifying results    comprises calling one of the nucleotide bases A, C, T or G.-   36. The device of any of clauses 34 to 35, wherein the logic to    classify results for two clusters includes mapping a first pixel    signal of the set of signal samples for a sampling event from a    particular sensor into at least four bins, and mapping a second    pixel signal of the set of signal samples for the sampling event    into at least four bins, and logically combining the mapping of the    first and second pixel signals to classify the results for two    clusters.-   37. The device of any of clauses 34 to 36, wherein the sample    surface holds clusters that are distributed unevenly over the pixel    areas, and the signal processor executes time sequence and spatial    analysis of the plurality of sequences of pixel signals to detect    patterns of illumination corresponding to individual clusters on the    sample surface, and to classify the results of the sampling events    for the individual clusters, wherein the plurality of sequences of    pixel signals encodes differential crosstalk between at least two    clusters resulting from their uneven distribution over the pixel    areas.-   38. The device of any of clauses 34 to 37, wherein the sample    surface comprises an array of wells overlying the pixel areas,    including two wells per pixel area, the two wells per pixel area    including a dominant well and a subordinate well, the dominant well    having a larger cross section over the pixel area than the    subordinate well.-   39. The device of any of clauses 34 to 38, wherein the sample    surface comprises an array of wells overlying the pixel areas, and    the sampling events include at least a first chemical stage with a    number K of illumination stages where K is a positive integer, where    the illumination stages of the K illumination stages illuminate the    pixel areas with different angles of illumination, and the sequences    of pixel signals include a set of signal samples for each sampling    event, the set including the number K of pixel signals for the at    least one chemical stage of the sampling events; wherein preferably    the sampling events further include a second chemical stage with a    number J of illumination stages where J is a positive integer, where    the illumination stages of the K illumination stages in the first    chemical stage and of the J illumination stages in the second    chemical stage illuminate the wells in the array of wells with    different angles of illumination, and the set of signal samples    further includes the number J of pixel signals for the second    chemical stage of the sampling events.-   40. The device of any of clauses 34 to 39, wherein the array of    sensors is included in a solid state imager.-   41. The device of any of clauses 34 to 40, wherein a pixel area    receives light from a well on the sample surface and the well is    configured to hold more than one cluster during the base calling    cycle, wherein a cluster preferably comprises a plurality of    single-stranded deoxyribonucleic acid (abbreviated DNA) fragments    having an identical nucleic acid sequence.-   42. A computer-implemented method of base calling, including:

for a base calling cycle of a sequencing by synthesis (SBS) run,receiving from a communication port

-   -   a plurality of sequences of pixel signals, the plurality of        sequences of pixel signals being generated, for a sequence of        sampling events comprising two illumination stages in time        sequence, based on light emitted by a plurality of clusters held        by a number N of pixel areas of a sample surface by an array of        sensors comprising light detectors, the array having a number N        of active sensors each associated with a corresponding pixel        area of the N pixel areas and configured to detect light        emissions gathered from the associated pixel area, the sensors        being configured to generate respective pixel signals during the        sequence of sampling events from the number N of corresponding        pixel areas of the sample surface to produce the plurality of        sequences of pixel signals, the sequences of pixel signals        including at least one pixel signal for each pixel area and        illumination stage, wherein at least one active sensor detects        light emissions from at least two clusters forming a cluster        pair of the plurality of clusters, wherein the intensity of        respective light emissions of the two clusters is significantly        different; and    -   processing the plurality of sequences of pixel signals to        classify results of the sequence of sampling events on clusters        in the plurality of clusters, including using the plurality of        sequences of pixel signals to classify results of the sequence        of sampling events on clusters in the plurality of clusters,        including using the plurality of sequences of pixel signals to        classify results of the sequence of sampling events on a number        N+M of clusters in the plurality of clusters from the number N        of active sensors, where Mis a positive integer, by classifying        results for the two clusters forming a cluster pair from the        sequences of pixel signals from the at least one active sensor        in the array of sensors.

-   43. The computer-implemented method of clause 42, further including:

mapping a first pixel signal, which represents light gathered from afirst pixel area during a first illumination stage of the base callingcycle, into at least four bins and mapping a second pixel signal, whichrepresents light gathered from the first pixel area during a secondillumination stage of the base calling cycle, into at least four bins,and

combining the mapping of the first and second pixel signals to identifythe incorporated nucleotide bases.

-   44. The computer-implemented method of any of clauses 42 to 43,    further including applying the method to identify the nucleotide    bases incorporated onto the plurality of clusters at a plurality of    pixel areas during the base calling cycle, preferably further    including repeating the method over successive base calling cycles    to identify the nucleotide bases incorporated onto the plurality of    clusters at the plurality of pixel areas during each of the base    calling cycles, more preferably further including:

for each of the base calling cycles, detecting and storing the first andsecond pixel signals emitted by the plurality of clusters at theplurality of pixel areas, and

after the base calling cycles, using the combination of the first andsecond pixel signals to identify the nucleotide bases incorporated ontothe plurality of clusters at the plurality of pixel areas during each ofthe previous base calling cycles.

-   45. The computer-implemented method of any of clauses 42 to 44,    wherein at least one of the following applies:

the first pixel area receives light from an associated well on a samplesurface; preferably light from more than one associated well on thesample surface;

the first and second pixel signals are gathered by a first sensor fromthe first pixel area, wherein the first and second pixel signals arepreferably detected by a signal processor configured for processingpixel signals gathered by the first sensor, and

the first illumination stage induces illumination from the first andsecond clusters to produce emissions from labeled nucleotide bases A andT and the second illumination stage induces illumination from the firstand second clusters to produce emissions from labeled nucleotide bases Cand T.

-   46. The computer-implemented method of any of clauses 42 to 45, in    which said base calling includes using a device as defined in any    one of clauses 34 to 41.-   47. A method of identifying pixel areas with more than one cluster    on a sample surface of a biosensor and base calling clusters at the    identified pixel areas, preferably the method of any of clauses    34-46, including:

performing a plurality of base calling cycles, each base calling cyclehaving a first illumination stage and a second illumination stage;

capturing at a sensor associated with a pixel area of the samplesurface,

-   -   a first set of intensity values generated during the first        illumination stage of the base calling cycles, and    -   a second set of intensity values generated during the second        illumination stage of the base calling cycles;

fitting sixteen distributions to the first and second sets of intensityvalues using a signal processor and, based on the fitting, classifyingthe pixel area as having more than one cluster; and

for a successive base calling cycle,

-   -   detecting the first and second sets of intensity values for a        cluster group at the pixel area using the signal processor, and    -   selecting a distribution for the cluster group, wherein the        distribution identifies a nucleotide base present in each        cluster of the cluster group

wherein preferably at least one of the following applies:

-   -   the fitting comprises using one or more algorithms, including a        k-means clustering algorithm, a k-means-like clustering        algorithm, an expectation maximization algorithm, and a        histogram based algorithm;    -   the method further comprises normalizing the intensity values;    -   the pixel area receives light from an associated well on the        sample surface; and    -   said identifying and base calling includes using a device as        defined in any one of clauses 34 to 41.

-   48. A computer-implemented method of base calling, preferably the    method according to any of clauses 42-46, the method comprising:

providing a first pixel signal that represents light gathered from afirst pixel area during a first illumination stage of a base callingcycle of a sequencing by synthesis (abbreviated SBS) run and a secondpixel signal that represents light gathered from said first pixel areaduring a second illumination stage of said base calling cycle of saidSBS run, wherein the first pixel area underlies first and secondclusters that share the first pixel area;

providing a signal processor configured for processing at least saidfirst and second pixel signals;

mapping the first pixel signal into at least four bins and mapping thesecond pixel signal into at least four bins using said signal processor;and

logically combining the mapping of the first and second pixel signals toidentify the nucleotide base incorporated onto each of said first andsecond clusters during said base calling cycle, or being a method ofidentifying pixel areas with more than one cluster on a sample surfaceof a biosensor and base calling clusters at the identified pixel areas,compnsmg:

-   -   providing a first set of intensity values generated during a        first illumination stage of a base calling cycle and a second        set of intensity values generated during a second illumination        stage of the base calling cycle, wherein the first and second        sets of intensity values represent the intensity of light        gathered at a sensor associated with a pixel area of the sample        surface;    -   fitting sixteen distributions to the first and second sets of        intensity values using a signal processor and, based on the        fitting, classifying the pixel area as having more than one        cluster; and    -   for a successive base calling cycle,        -   providing first and second sets of intensity values for a            cluster group at the pixel area using the signal processor,            and        -   selecting a distribution for the cluster group, wherein the            distribution identifies a nucleotide base present in each            cluster of the cluster group, wherein the fitting preferably            comprises using one or more algorithms, including a k-means            clustering algorithm, a k-means-like clustering algorithm,            an expectation maximization algorithm, and a histogram based            algorithm.

What is claimed is:
 1. A device for base calling, comprising: areceptacle configured to hold a biosensor, the biosensor having: asample surface including pixel areas and holding a plurality of clustersduring a sequence of sampling events such that the clusters aredistributed unevenly over the pixel areas., each of the clustersincludes a plurality of oligonucleotides; an array of sensors configuredto generate a plurality of sequences of pixel signals, the array havinga number N of active sensors, the sensors in the array disposed relativeto the sample surface to generate respective pixel signals during thesequence of sampling events from the number N of corresponding pixelareas of the sample surface to produce the plurality of sequences ofpixel signals; and a communication port which outputs the plurality ofsequences of pixel signals; and a signal processor coupled to thereceptacle, and configured to execute time sequence and spatial analysisof the plurality of sequences of pixel signals to detect patterns ofillumination corresponding to a number N+M of individual clusters on thesample surface from the number N of active sensors, where M is apositive integer, and to disaggregate signals in at least one of thesequences of pixel signals that mix two clusters and to classify theresults of the sequence of sampling events for the number N+M ofindividual clusters.
 2. The device of claim 1, wherein the signalprocessor uses the detected patterns of illumination to locate thenumber N+M of individual clusters on the sample surface from the numberN of active sensors.
 3. A device for base calling, comprising: abiosensor having a sample surface including pixel areas and an array ofwells overlying the pixel areas, including two wells per pixel area, thetwo wells including a dominant well and a distinct subordinate well, thedominant well having a larger cross section over the pixel area than thesubordinate well, wherein a cluster of oligonucleotides is immobilizedin each of the two wells, and immobilized clusters in the dominant welland the subordinate well contribute to a combined signal.
 4. The deviceof claim 3, wherein the biosensor comprises: an array of sensors, whereeach sensor in the array senses information from the two clustersdisposed in corresponding pixel areas of the sample surface to generatea pixel signal in each sampling event, the array configured to generatea plurality of sequences of pixel signals, the array having a number Nof active sensors, the sensors in the array disposed relative to thesample surface to generate respective pixel signals during the sequenceof sampling events from the number N of corresponding pixel areas of thesample surface to produce the plurality of sequences of pixel signals,and a communication port which outputs the plurality of sequences ofpixel signals.
 5. The device of claim 3, wherein the two wells havedifferent offsets relative to a center of the pixel area.
 6. The deviceof claim 5, wherein, during each sampling event, the pixel area receivesdifferent amounts of illumination from the two wells
 7. The device ofclaim 6, wherein each of the two wells holds at least one cluster duringeach sampling event.
 8. The device of claim 7, wherein, during eachsampling event, the pixel area receives an amount of illumination from abright cluster in the dominant well that is greater than an amount ofillumination received from a dim cluster in the subordinate well.
 9. Thedevice of claim 8, wherein the biosensor is coupled to a signalprocessor, the signal processor configured to receive and to process theplurality of sequences of pixel signals to identify bases present in anumber N+M of clusters from the number N of active sensors, includingfor the bright and dim cluster: mapping into at least four bins a firstpixel signal generated by a sensor corresponding to the pixel areaduring a first illumination stage of each sampling event, mapping intoat least four bins a second pixel signal generated by the sensor duringa second illumination stage of each sampling event, and logicallycombining the mapping of the first and second pixel signals to identifythe bases present in the bright cluster and the dim cluster.
 10. Adevice for base calling, comprising: a biosensor having a sample surfaceincluding pixel areas and an array of wells overlying the pixel areas;and an illumination system that is coupled to the sample surface andilluminates the pixel areas with different angles of illumination duringa sequence of sampling events, including, for each sampling event in thesequence of sampling events, illuminating each of the wells withoff-axis illumination to produce asymmetrically illuminated well regionsin each of the wells
 11. The device of claim 10, wherein the biosensorincludes an array of sensors configured to generate a plurality ofsequences of pixel signals, the array having a number N of activesensors, the sensors in the array disposed relative to the samplesurface to generate respective pixel signals during the sequence ofsampling events from the number N of corresponding pixel areas of thesample surface to produce the plurality of sequences of pixel signals,and a communication port which outputs the plurality of sequences ofpixel signals.
 12. The device of claim 10, wherein the asymmetricallyilluminated regions of a well include at least a dominant well regionand a subordinate well region, such that during each sampling event thedominant well region is illuminated more than the subordinate wellregion.
 13. The device of claim 12, wherein the dominant and subordinatewell regions each includes a cluster.
 14. The device of claim 13,wherein, during each sampling event, a pixel area overlying the wellreceives an amount of illumination from a bright cluster in the dominantwell region that is greater than an amount of illumination received froma dim cluster in the subordinate well region.
 15. The device of claim14, wherein the biosensor is coupled to a signal processor, the signalprocessor configured to receive and to process the plurality ofsequences of pixel signals to identify bases present in a number N+M ofclusters from the number N of active sensors, including for the brightand dim cluster: mapping into at least four bins a first pixel signalgenerated by a sensor corresponding to the pixel area during a firstillumination stage of each sampling event, mapping into at least fourbins a second pixel signal generated by the sensor during a secondillumination stage of each sampling event, and logically combining themapping of the first and second pixel signals to identify the basespresent in the bright cluster and the dim cluster.
 16. The device ofclaim 10, wherein the off-axis illumination is at a forty-five degreeangle.
 17. The device of claim 10, wherein one well of the array ofwells overlies per pixel area.
 18. The device of claim 10, wherein twowells of the array of wells overlie per pixel area.